Soybean 1-deoxy-D-xylulose 5-phosphae synthase and DNA encoding thereof

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding an isopentenyl diphosphate biosynthetic enzyme. The invention also relates to the construction of a chimeric gene encoding all or a portion of the isopentenyl diphosphate biosynthetic enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the isopentenyl diphosphate biosynthetic enzyme in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No. 60/110,779, filed Dec. 3, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding 1-deoxy-D-xylulose 5-phosphate synthase in plants and seeds.

BACKGROUND OF THE INVENTION

Isoprenoids comprise the largest family of natural products, including numerous secondary compounds, which play different functional roles in plants such as hormones, photosynthetic pigments, electron carriers, and structural components of membranes. The fundamental unit in isoprenoid biosynthesis, isopentenyl diphosphate (IPP), is normally synthesized by the condensation of acetyl CoA through the mevalonate pathway. In many organisms including several bacteria, algae and plant plastids, IPP is synthesized by a mevalonate-independent pathway. The initial step, in this pathway is the condensation of pyruvate and glyceraldehyde 3-phosphate to form 1-deoxy-D-xylulose 4-phosphate which behaves as the precursor for IPP, thiamine (vitamin B1), or pyridoxine (vitamin B2). This initial step is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), a member of a distinct proteins family. In E. coli DXPS shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase (Sprenger (1997) Proc. Natl. Acad. Sci. USA 94:12857-12862).

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 170 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NOs:2, 4, 18, 20, 22, and 24, a rice 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NOs:6, 8, 26, and 28, a soybean 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NOs:10 and 12, a wheat 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of SEQ ID NO:14, 16, 30, and 32. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

It is preferred that the isolated polynucleotide of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and the complement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.

The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.

The present invention relates to a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide of at least 170 amino acids comprising at least 95% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in a host cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in the host cell containing the isolated polynucleotide with the level of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide in a.host cell that does not contain the isolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide gene, preferably a plant 1-deoxy-D-xylulose 5-phosphate synthase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a 1-deoxy-D-xylulose 5-phosphate synthase amino acid sequence.

The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a 1-deoxy-D-xylulose 5-phosphate synthase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a 1-deoxy-D-xylulose 5-phosphate synthase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a 1-deoxy-D-xylulose 5-phosphate synthase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric.gene results in production of 1-deoxy-D-xylulose 5-phosphate synthase in the transformed host cell; (c) optionally purifying the 1-deoxy-D-xylulose 5-phosphate synthase expressed by the transformed host cell; (d) treating the 1-deoxy-D-xylulose 5-phosphate synthase with a compound to be tested; and (e) comparing the activity of the 1-deoxy-D-xylulose 5-phosphate synthase that has been treated with a test compound to the activity of an untreated 1-deoxy-D-xylulose 5-phosphate synthase, thereby selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWING AND SEOUENCE DESCRIPTIONS

The invention can be more fully understood from the following detailed description and the accompanying drawing and Sequence Listing which form a part of this application.

FIGS. 1A-D shows a comparison of the amino acid sequences of the 1-deoxy-D-xylulose 5-phosphate synthase from soybean clone sdp2c.pk00.h19 (SEQ ID NO:10), soybean clone sgc1c.pk001.c11 (SEQ ID NO:12), rice clone rl0n.pk081.m14 (SEQ ID NO:26), Capsicum annuum set forth in NCBI General Identifier No. 3559816 (SEQ ID NO:33), and Oryza sativa set forth in NCBI General Identifier No. 3913239 (SEQ ID NO:34). Amino acids conserved among all sequences are indicated with an asterisk (*) on the top row; dashes are used by the program to maximize alignment of the sequences.

Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

TABLE 1 Isopentenyl Diphosphate Biosynthetic Enzymes SEQ ID NO: (Nucleo- (Amino Protein Clone Designation tide) Acid) Corn 1-Deoxy-D-Xylulose Contig of: 1 2 5-Phosphate Synthase csi1n.pk0040.e11 csi1n.pk0043.b2 cen5.pk0058.b3 p0014.ctuse54r Corn 1-Deoxy-D-Xylulose p0006.cbyvq72r 3 4 5-Phosphate Synthase Rice 1-Deoxy-D-Xylulose Contig of: 5 6 5-Phosphate Synthase r10n.pk081.m14 r1r24.pk0087.h4 Rice 1-Deoxy-D-Xylulose rr1.pk089.113 7 8 5-Phosphate Synthase Soybean 1-Deoxy-D-Xylulose sdp2c.pk001.h19 9 10 5-Phosphate Synthase Soybean 1-Deoxy-D-Xylulose sgc1c.pk001.c11 11 12 5-Phosphate Synthase Wheat 1-Deoxy-D-Xylulose w1m4.pk0022.h2 13 14 5-Phosphate Synthase Wheat 1-Deoxy-D-Xylulose w1m4.pk0009.c9 15 16 5-Phosphate Synthase Corn 1-Deoxy-D-Xylulose Contig of: 17 18 5-Phosphate Synthase cen5.pk0058.b3:fis csi1n.pk0040.e11 Corn 1-Deoxy-D-Xylulose p0006.cbyvq72r:fis 19 20 5-Phosphate Synthase Corn 1-Deoxy-D-Xylulose p0031.ccmcg27ra 21 22 5-Phosphate Synthase Corn 1-Deoxy-D-Xylulose p0126.cn1cx46r 23 24 5-Phosphate Synthase Rice 1-Deoxy-D-Xylulose r10n.pk081.m14:fis 25 26 5-Phosphate Synthase Rice 1-Deoxy-D-Xylulose rr1.pk089.113:fis 27 28 5-Phosphate Synthase Wheat 1-Deoxy-D-Xylulose w1m4.pk0009.c9 29 30 5-Phosphate Synthase Wheat 1-Deoxy-D-Xylulose w1m4.pk0022.h2 31 32 5-Phosphate Synthase

The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized. As used herein, a “polynucleotide” is a nucleotide sequence such as a nucleic acid fragment. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA. An isolated polynucleotide of the present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides, of the nucleic acid sequence of the SEQ. ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such sequences.

As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example, antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a polypeptide in a plant cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial, or viral) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measurng the level a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1 985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with. 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least about 50 amino acids, preferably at least about 100 amino acids, more preferably at least about 150 amino acids, still more preferably at least about 200 amino acids, and most preferably at least about 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED-5.

A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following. (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature; “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptide by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme, RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

“Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050, incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at leasta portion of several 1-deoxy-D-xylulose 5-phosphate synthases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other 1-deoxy-D-xylulose 5-phosphate synthases, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide. The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide of a gene (such as 1-deoxy-D-xylulose 5-phosphate synthase) preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a polypeptide.

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptide is present at higher or lower levels than normal or in cell types or developmental stages in which it is not normally found. This would have the effect of altering the level of isopentenyl diphosphate in those cells. Manipulation of this gene in the endosperm of plants could result in increased xanthophyll levels, which has value as coloring agents in poultry feeds. In Arabidopsis, mutants in this gene are carotenoid deficient and albino. Because this mevalonate-independent pathway appears to be unique to microorganisms and plastids inhibitors of this enzyme should have no affect on animals. Overexpression of this gene will produce the active enzyme for high-through screening to find inhibitors for this enzyme. These inhibitors may lead to discover a novel herbicide.

Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. For reasons of convenience, the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

For some applications it may be useful to direct the instant polypeptide to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by altering the coding sequence to encode the instant polypeptide with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds, and is not an inherent part of the invention. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

The instant polypeptide (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptide of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptide are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptide. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded 1-deoxy-D-xylulose 5-phosphate synthase. An example of a vector for high level expression of the instant polypeptide in a bacterial host is provided (Example 6).

Additionally, the instant polypeptide can be used as a target to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptide described herein catalyzes isopentenyl diphosphate synthesis via the mevalonate-independent pathway. Accordingly, inhibition of the activity of the enzyme described herein could lead to inhibition of plant growth. Thus, the instant 1-deoxy-D-xylulose 5-phosphate synthase could be appropriate for new herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:956-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation -Hybrid Mapping (Walter et al. ((1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptide. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptide can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptide disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean, and wheat tissues were prepared. The characteristics of the libraries are described below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat Library Tissue Clone cen5 Corn Endosperm 30 Days After Pollination cen5.pk0058.b3 csi1n Corn Silk* csi1n.pk0040.e11 csi1n Corn Silk* csi1n.pk0043.b2 p0006 Corn, Young Shoot p0006.cbyvq72r p0014 Corn Leaves 7 and 8 from Plant Transformed p0014.ctuse54r with uaz151 (G-protein) Gene, C. heterostrophus Resistant p0031 Corn Shoot Culture p0031.ccmcg27ra p0126 Corn Leaf Tissue Pooled From V8-V10 p0126.cn1cx46r Stages**, Night-Harvested r10n Rice 15 Day Old Leaf* r10n.pk081.m14 r1r24 Rice Leaf 15 Days After Germination, 24 r1r24.pk0087.h4 Hours After Infection of Strain Magaporthe grisea 4360-R-62 (AVR2- YAMO); Resistant rr1 Rice Root of Two Week Old Developing rr1.pk089.113 Seedling sdp2c Soybean Developing Pods (6-7 mm) sdp2c.pk001.h19 sgc1c Soybean Cotyledon 7 Days After sgc1c.pk001.c11 Germination (Young Green) w1m4 Wheat Seedlings 4 Hours After Inoculation w1m4.pk0009.c9 With Erysiphe graminis f. sp tritici w1m4 Wheat Seedlings 4 Hours After Inoculation w1m4.pk0022.h2 With Erysiphe graminis f. sp tritici *These libraries were normalized essentially as described in U.S. Pat. No. 5,482,845, incorporated herein by reference. **Corn developmental stages are explained in the publication “How a corn plant develops” from the Iowa State University Coop. Ext. Service Special Report No. 48 reprinted June 1993.

cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding 1-deoxy-D-xylulose 5-phosphate synthases were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as “pLog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding 1-Deoxy-D-Xylulose 5-Phosphate Synthase

The BLASTX search using the nucleotide sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to 1-deoxy-D-xylulose 5-phosphate synthase from Capsicum annuum (NCBI General Identifier No. 3559816). Shown in Table 3 are the BLAST results for individual ESTs (“EST”), contigs assembled from two or more ESTs (“Contig”), or sequences encoding the entire protein derived from the entire cDNA inserts comprising the indicated cDNA clones (FIS), a contig, or an FIS and PCR (“CGS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to 1-Deoxy-D-Xylulose 5-Phosphate Synthase BLAST pLog Score Clone Status 3559816 Contig of: Contig 84.70 csi1n.pk0040.e11 csi1n.pk0043.b2 cen5.pk0058.b3 p0014.ctuse54r p0006.cbyvq72r EST 66.40 Contig of: Contig 18.00 r10n.pk081.m14 r1r24.pk0087.h4 rr1.pk089.113 EST 25.30 sdp2c.pk001.h19 CGS >254 sgc1c.pk001.c11 CGS >254 w1m4.pk0022.h2 EST 16.00 w1m4.pk0009.c9 EST 62.00

Further sequencing of some of the above clones yielded new information. The BLASTX search using the nucleotide sequences from clones listed in Table 4 revealed similarity of the polypeptides encoded by the cDNAs to transketolase 2 from Oryza sativa and Capsicum annuum (NCBI General Identifier Nos. 5803266 and 3559816, respectively), and 1-deoxy-D-xylulose 5-phosphate synthase from Oryza sativa, Lycopersicon esculentum, and Catharanthus roseus (NCBI General Identifier Nos. 3913239, 5059160, and 3724087, respectively). Shown in Table 4 are the BLAST results for individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from an FIS and an EST (“Contig*”), or sequences encoding the entire protein derived from an FIS, or an FIS and PCR (“CGS”):

TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous to 1-Deoxy-D-Xylulose 5-Phosphate Synthase NCBI General Clone Status Identifier No. BLAST pLog Score Contig of: Contig* 5803266 140.00 cen5.pk0058.b3:fis csi1n.pk0040.e11 p0006.cbyvq72r:fis FIS 5059160 >254.00 p0031.ccmcg27ra EST 3724087 68.52 p0126.cn1cx46r EST 5803266 32.05 r10n.pk081.m14:fis FIS 3913239 >254.00 rr1.pk089.113:fis CGS 3559816 >254.00 w1m4.pk0009.c9 EST 3559816 91.52 w1m4.pk0022.h2 Contig 3913239 >254.00

FIGS. 1A-D presents an alignment of the amino acid sequences set forth in SEQ ID NOs:10, 12, and 26 and the Capsicum annuum and Oryza sativa sequences (SEQ ID NO:33 and SEQ ID NO:34, respectively). The data in Table 5 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 and the Capsicum annuum sequence (SEQ ID NO:33).

TABLE 5 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to 1-Deoxy-D-Xylulose 5-Phosphate Synthase Percent Identity to SEQ ID NO. 3559816 2 61.6 4 86.3 6 37.6 8 66.7 10 87.2 12 86.6 14 38.7 16 80.6 18 60.7 20 86.9 22 62.7 24 45.7 26 82.5 28 55.9 30* 77.6 32* 75.1 *SEQ ID NO:30 encodes the C-terminal fourth of a wheat 1-deoxy-D-xylulose 5-phosphate synthase while SEQ ID NO:32 encodes the N-terminal third of the protein

Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of corn, rice, and wheat and entire soybean and rice 1-deoxy-D-xylulose 5-phosphate synthases. There are at least two independent 1-deoxy-D-xylulose 5-phosphate synthase variants in each crop. These sequences represent the first corn, soybean, and wheat sequences encoding 1-deoxy-D-xylulose 5-phosphate synthase and variants of rice 1-deoxy-D-xylulose 5-phosphate synthase.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptide in sense orientation with respect to the maize 27 kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that is located 3′ to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then, digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML 103. Plasmid pML 103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5′ to 3′direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the.dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfifrt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium lumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supenatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuiged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Biol/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptide in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed. expression cassette.

Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptide. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 2,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. 1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptide and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each tnansformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post. bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos. Example 6

Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptide can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, was converted to 5-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaser™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be.selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final, concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 7 Evaluating Compounds for Their Ability to Inhibit the Activity of Isopentenyl Diphosnhate Biosynthetic Enzymes

The polypeptide described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 6, or expression in eukaryotic cell culture, in planta, and using viral expression systems in suitably infected organisms or cell lines. The instant polypeptide may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase (“GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide (“(His)₆”). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant polypeptide, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the instant polypeptide are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, the instant polypeptide may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)₆ peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin.

Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation of the instant polypeptide disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. For example, assays for 1-deoxy-D-xylulose 5-phosphate synthase are presented by Sprenger et al. (1998) Proc. Natl. Acad. Sci. USA 95:2105-2110).

34 1 738 DNA Zea mays unsure (634) n = A, C, G or T 1 gagaatgaca agcgcattgt ggtagttcat ggcggcatgg gaatcgatcg atcactccgc 60 ttattccagt ccaggttccc agacagattt tttgacttgg gcatcgctga gcaacatgct 120 gttacctttt ctgctggttt ggcctgcgga ggtctaaagc ctttctgcat aattccatcc 180 acatttcttc agcgagcata tgatcagata attgaagatg tggacatgca aaagatacca 240 gttcgttttg ctatcacaaa tgctggtctg gtaggatctg agggtccaac taattcagga 300 ccatttgata ttacattcat gtcatgcttg ccaaacatga ttgtcatgtc accatctaat 360 gaggatgaac ttattgacat ggtggcaaca gctgcaatga ttgaggacag acctatttgc 420 ttccgctatc ctaggggtgc cattgttggg actagtggaa gtgtaacata tgggaatcca 480 tttgagattg gtaaaggaga gattcttgtc gagggaaaag agatagcttt tcttggctat 540 ggcgaggtgg tccagagatg cttgattgct cgatcccttt tatccaactt gggcattcag 600 gcgacagttg caaatgcgag gttttgcaag ccgntntgac agcggaccta atccagaacg 660 ntgttgccag cncatgagtt tttcttgacc acagnggaaa gaaaggaacg gtntggnagg 720 gctttgggag caacaggt 738 2 211 PRT Zea mays 2 Glu Asn Asp Lys Arg Ile Val Val Val His Gly Gly Met Gly Ile Asp 1 5 10 15 Arg Ser Leu Arg Leu Phe Gln Ser Arg Phe Pro Asp Arg Phe Phe Asp 20 25 30 Leu Gly Ile Ala Glu Gln His Ala Val Thr Phe Ser Ala Gly Leu Ala 35 40 45 Cys Gly Gly Leu Lys Pro Phe Cys Ile Ile Pro Ser Thr Phe Leu Gln 50 55 60 Arg Ala Tyr Asp Gln Ile Ile Glu Asp Val Asp Met Gln Lys Ile Pro 65 70 75 80 Val Arg Phe Ala Ile Thr Asn Ala Gly Leu Val Gly Ser Glu Gly Pro 85 90 95 Thr Asn Ser Gly Pro Phe Asp Ile Thr Phe Met Ser Cys Leu Pro Asn 100 105 110 Met Ile Val Met Ser Pro Ser Asn Glu Asp Glu Leu Ile Asp Met Val 115 120 125 Ala Thr Ala Ala Met Ile Glu Asp Arg Pro Ile Cys Phe Arg Tyr Pro 130 135 140 Arg Gly Ala Ile Val Gly Thr Ser Gly Ser Val Thr Tyr Gly Asn Pro 145 150 155 160 Phe Glu Ile Gly Lys Gly Glu Ile Leu Val Glu Gly Lys Glu Ile Ala 165 170 175 Phe Leu Gly Tyr Gly Glu Val Val Gln Arg Cys Leu Ile Ala Arg Ser 180 185 190 Leu Leu Ser Asn Leu Gly Ile Gln Ala Thr Val Ala Asn Ala Arg Phe 195 200 205 Cys Lys Pro 210 3 415 DNA Zea mays unsure (11)..(13) n = A, C, G or T 3 agcagatgga nnnactcagt gcacgagctg gcggcgaagt ggacgagtac gcccgcggca 60 tgatcagcgg gcccggctcc tcgctcttcg aggagctcgg tctctactac atcggccccg 120 tcgacggcca caacatcgac gacctcatca ccatcctcaa cgacgtcaag agcaccaaga 180 ccaccggccc cgtcctcatc cacgtcgtca ccgagaaggg ccgcggctac ccctacgccg 240 agcgagccgc cgacaagtac cacggtgtcg ccaagtttga tccggcgacc gggaagcagt 300 tcaagtcccc cgccaagacg ctgtcctaca ccaactactt cgccgaggcg ctcatcgccg 360 aggcggagca ggacagcaag atcgtnggca tccangcggc catgggggcg gacgg 415 4 131 PRT Zea mays UNSURE (127) Xaa = ANY AMINO ACID 4 Ser Val His Glu Leu Ala Ala Glu Val Asp Glu Tyr Ala Arg Gly Met 1 5 10 15 Ile Ser Gly Pro Gly Ser Ser Leu Phe Glu Glu Leu Gly Leu Tyr Tyr 20 25 30 Ile Gly Pro Val Asp Gly His Asn Ile Asp Asp Leu Ile Thr Ile Leu 35 40 45 Asn Asp Val Lys Ser Thr Lys Thr Thr Gly Pro Val Leu Ile His Val 50 55 60 Val Thr Glu Lys Gly Arg Gly Tyr Pro Tyr Ala Glu Arg Ala Ala Asp 65 70 75 80 Lys Tyr His Gly Val Ala Lys Phe Asp Pro Ala Thr Gly Lys Gln Phe 85 90 95 Lys Ser Pro Ala Lys Thr Leu Ser Tyr Thr Asn Tyr Phe Ala Glu Ala 100 105 110 Leu Ile Ala Glu Ala Glu Gln Asp Ser Lys Ile Val Gly Ile Xaa Ala 115 120 125 Ala Met Gly 130 5 717 DNA Oryza sativa unsure (581) n = A, C, G or T 5 cttacatgtc ctttctccac ctcggtggtc atcagctaga cagctatcgc gcgccgtccc 60 accaccatct tgctccacta ccgcggacca ccgcgcgcga gcagagcatc tcctcactct 120 ctagcttgct ccagtttcgc gtagctgcgt gacagttcaa ttgaactctc tggattcgtt 180 ggttacttcg tctgagctgc tgcagcgttg aggaggagga ggagcaatgg cgctcacgac 240 gttctccatt tcgagaggag gcttcgtcgg cgcgctgccg caggaggggc atttcgctcc 300 ggcggcggcg gagctcagtc tccacaagct ccagagcagg ccacacaagg ctaggcggag 360 gtcgtccgtc gagcatctcg gcgtcgctgt ccacgggaga gggaggcggc ggatacaatc 420 gcaagcggca ccgacgccgc tgctggacac gtcaaactac cccatccaca tgaaagaact 480 gtccctcaaa ggactccagc aactcgccga cgagctcgct ccgactcatc ctcactctcc 540 aaagaccggg ggacatctcg ggtccaacct cggcgtcgtc naactcaccg tcgcgctcca 600 ctaactgttc aacaccctca ggacaagatc tctgggactc ggcacaatcg tacctcacaa 660 aantctgacg ggcggngcga caagatncga caagcgtaga caacggttnt cggaatc 717 6 125 PRT Oryza sativa UNSURE (119) Xaa = ANY AMINO ACID 6 Met Ala Leu Thr Thr Phe Ser Ile Ser Arg Gly Gly Phe Val Gly Ala 1 5 10 15 Leu Pro Gln Glu Gly His Phe Ala Pro Ala Ala Ala Glu Leu Ser Leu 20 25 30 His Lys Leu Gln Ser Arg Pro His Lys Ala Arg Arg Arg Ser Ser Val 35 40 45 Glu His Leu Gly Val Ala Val His Gly Arg Gly Arg Arg Arg Ile Gln 50 55 60 Ser Gln Ala Ala Pro Thr Pro Leu Leu Asp Thr Ser Asn Tyr Pro Ile 65 70 75 80 His Met Lys Glu Leu Ser Leu Lys Gly Leu Gln Gln Leu Ala Asp Glu 85 90 95 Leu Ala Pro Thr His Pro His Ser Pro Lys Thr Gly Gly His Leu Gly 100 105 110 Ser Asn Leu Gly Val Val Xaa Leu Thr Val Ala Leu His 115 120 125 7 494 DNA Oryza sativa unsure (19) n = A, C, G or T 7 acatacatat gcacacaana ttctcacang aaggggctca ctcnttcata ctattaagca 60 aagaaagggg ntttcangtt tcacatcccg tttcnagagc gaatatgatn cctttggtgc 120 angacatgga tgcaataatc tctccncaag ccttgggatg gcantcncaa gggatctaag 180 tgggaggaaa aaccnaatag taacagttat aagtaactgg acaactatgg ctggtcangt 240 gtatgaggca atgggtcatg ccggtttcct tgattctaac atggnagtga ttttaaatga 300 caagccggga caccttgctt cctaaagcan atagccaatc aaagatgtct attaatgccc 360 tcncnaatgc tctgagcaaa gntcaatcca acaaaaggat ttataaagtt taagganggt 420 gcaaaaggga ctttccaaat ggttttggta aaaggaagcn atgaanttnc tgccaaaaat 480 tattaatatg cccc 494 8 21 PRT Oryza sativa UNSURE (15) Xaa = ANY AMINO ACID 8 Val Tyr Glu Ala Met Gly His Ala Gly Phe Leu Asp Ser Asn Xaa Met 1 5 10 15 Val Ile Leu Asn Asp 20 9 2583 DNA Glycine max 9 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctccacc 60 gcggtggcgg ccgctctaga actagtggat cccccgggct gcaggaattc ggcacgaggt 120 gaagttcacc ttgttcctca caataattct ctcctacctc ttgtgttttg cttcagtcat 180 gtctctctct gcattctcat tccctctcca tctgagacaa acaacaccac cttctgatcc 240 taaaacatca tcaacccctt tgcctttgtc ttctcactcc cattggggtg cagatctgct 300 cacacaatcc caacgcaaac tcaaccaggt gaaaagaagg ccacatgggg tatgtgcatc 360 actatcagaa atgggggagt attattctca gaaacctcct actccactgt tggacaccat 420 aaactatcca attcacatga agaatttggc taccaagaaa ctgaaacaac ttgcggatga 480 gctgcgttct gatgttattt tccatgtttc tagaactggg ggtcatttgg gatctagcct 540 tggtgttgta gaactcacta ttgcccttca ctatgttttc aatgctccta aggacaaaat 600 tttgtgggat gttggtcatc agtcttatcc tcataagata ctcactggta gaagggataa 660 gatgcatacc atgaggcaga cagatggatt ggccgggttt acaaaacgat ctgagagtga 720 ttatgattgt tttggcactg gtcacagctc cacaacaata tcagcaggac tgggaatggc 780 tgttgggagg gatctgaagg gagacaagaa taatgtagtt gctgttatcg gtgatggtgc 840 tatgacggct ggtcaagctt atgaagccat gaacaacgct ggatatcttg attccgacat 900 gattgttatt ctaaatgaca acaagcaggt ctccctacca actgctaatc tcgatggtcc 960 cataccacct gtaggtgctt tgagtagtgc tctcagtaag ttacaatcaa acagacctct 1020 tagagaactc agagaggttg ctaagggagt cactaaacaa attggtggcc caatgcatga 1080 gttagctgca aaagttgatg aatatgcgcg tggcatgatc agcggttctg gatcaacact 1140 atttgaagag cttggacttt actacatagg tcctgttgat ggtcataata tagatgatct 1200 tgtgtccatt ctaaatgaag ttaaaagtac taaaacaact ggtcctgtgc tgctccatgt 1260 tgtcactgaa aaaggccatg gatatccata tgcagaaaga gcagcagaca agtaccatgg 1320 agttactaag tttgatccag caactggaaa acaattcaaa tccaatgctg ccacccagtc 1380 atacacaaca tactttgcag aggctttaat tgctgaagcg gaagctgaca aagacattgt 1440 cggaatccat gctgcaatgg gaggtggaac tggcatgaat ctcttccttc gccgtttccc 1500 aacaagatgc tttgatgtgg ggatagcaga acagcatgct gttacatttg cggctggtct 1560 ggcttgtgaa ggccttaagc ctttttgtgc aatttactca tcatttatgc agagagctta 1620 tgaccaggtg gtgcatgatg tcgatttgca gaagctgcct gtaagattcg caatggaccg 1680 agccggatta gttggagcag atggtcccac acactgcggt gcatttgatg tcacttttat 1740 ggcatgcctc cctaacatgg tggtgatggc tccttctgat gaagcagagc tttttcacat 1800 ggttgcaact gcagctgcca ttgatgatcg acccagttgt ttccgatacc cgaggggaaa 1860 tggtattggt gttgagctac cactagggaa taaaggcatt cctcttgaga ttgggaaggg 1920 taggatacta attgaaggag aaagagtggc cttgttgggc tatggatcag ctgttcagag 1980 ctgtctggct gctgcttcct tgttggaaca tcatggcttg cgcgcaacag tggcggatgc 2040 acgtttctgc aagccattgg accgttccct tattcgcagc cttgcccaat cgcacgaggt 2100 tttgatcact gtggaagaag ggtcaatagg aggattcgga tctcatgttg ttcagttcat 2160 ggcccttgat ggccttcttg atgggaaatt aaagtggagg ccaattgttc ttcctgattg 2220 ttacattgac catggatcac cggttgacca attgagtgca gctggtctta caccatctca 2280 catagcagca acagttttca atctacttgg acaaacaaga gaggcactag aggtcatgac 2340 ataaaacaaa tgcaaagggg ttcaattttt gttccctgca atgtacaaag tagcgtgatt 2400 cacccagtgt aatacaaatg tgtttgttaa aaaataatag aaatggaaaa tgcagattga 2460 caaataatag tgccaacaaa tggttaaacg aataaaaaaa aaaaaaaaaa actcgagggg 2520 gggcccggta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 2580 ttt 2583 10 721 PRT Glycine max 10 Met Ser Leu Ser Ala Phe Ser Phe Pro Leu His Leu Arg Gln Thr Thr 1 5 10 15 Pro Pro Ser Asp Pro Lys Thr Ser Ser Thr Pro Leu Pro Leu Ser Ser 20 25 30 His Ser His Trp Gly Ala Asp Leu Leu Thr Gln Ser Gln Arg Lys Leu 35 40 45 Asn Gln Val Lys Arg Arg Pro His Gly Val Cys Ala Ser Leu Ser Glu 50 55 60 Met Gly Glu Tyr Tyr Ser Gln Lys Pro Pro Thr Pro Leu Leu Asp Thr 65 70 75 80 Ile Asn Tyr Pro Ile His Met Lys Asn Leu Ala Thr Lys Lys Leu Lys 85 90 95 Gln Leu Ala Asp Glu Leu Arg Ser Asp Val Ile Phe His Val Ser Arg 100 105 110 Thr Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu Leu Thr Ile 115 120 125 Ala Leu His Tyr Val Phe Asn Ala Pro Lys Asp Lys Ile Leu Trp Asp 130 135 140 Val Gly His Gln Ser Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp 145 150 155 160 Lys Met His Thr Met Arg Gln Thr Asp Gly Leu Ala Gly Phe Thr Lys 165 170 175 Arg Ser Glu Ser Asp Tyr Asp Cys Phe Gly Thr Gly His Ser Ser Thr 180 185 190 Thr Ile Ser Ala Gly Leu Gly Met Ala Val Gly Arg Asp Leu Lys Gly 195 200 205 Asp Lys Asn Asn Val Val Ala Val Ile Gly Asp Gly Ala Met Thr Ala 210 215 220 Gly Gln Ala Tyr Glu Ala Met Asn Asn Ala Gly Tyr Leu Asp Ser Asp 225 230 235 240 Met Ile Val Ile Leu Asn Asp Asn Lys Gln Val Ser Leu Pro Thr Ala 245 250 255 Asn Leu Asp Gly Pro Ile Pro Pro Val Gly Ala Leu Ser Ser Ala Leu 260 265 270 Ser Lys Leu Gln Ser Asn Arg Pro Leu Arg Glu Leu Arg Glu Val Ala 275 280 285 Lys Gly Val Thr Lys Gln Ile Gly Gly Pro Met His Glu Leu Ala Ala 290 295 300 Lys Val Asp Glu Tyr Ala Arg Gly Met Ile Ser Gly Ser Gly Ser Thr 305 310 315 320 Leu Phe Glu Glu Leu Gly Leu Tyr Tyr Ile Gly Pro Val Asp Gly His 325 330 335 Asn Ile Asp Asp Leu Val Ser Ile Leu Asn Glu Val Lys Ser Thr Lys 340 345 350 Thr Thr Gly Pro Val Leu Leu His Val Val Thr Glu Lys Gly His Gly 355 360 365 Tyr Pro Tyr Ala Glu Arg Ala Ala Asp Lys Tyr His Gly Val Thr Lys 370 375 380 Phe Asp Pro Ala Thr Gly Lys Gln Phe Lys Ser Asn Ala Ala Thr Gln 385 390 395 400 Ser Tyr Thr Thr Tyr Phe Ala Glu Ala Leu Ile Ala Glu Ala Glu Ala 405 410 415 Asp Lys Asp Ile Val Gly Ile His Ala Ala Met Gly Gly Gly Thr Gly 420 425 430 Met Asn Leu Phe Leu Arg Arg Phe Pro Thr Arg Cys Phe Asp Val Gly 435 440 445 Ile Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Cys Glu 450 455 460 Gly Leu Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Met Gln Arg Ala 465 470 475 480 Tyr Asp Gln Val Val His Asp Val Asp Leu Gln Lys Leu Pro Val Arg 485 490 495 Phe Ala Met Asp Arg Ala Gly Leu Val Gly Ala Asp Gly Pro Thr His 500 505 510 Cys Gly Ala Phe Asp Val Thr Phe Met Ala Cys Leu Pro Asn Met Val 515 520 525 Val Met Ala Pro Ser Asp Glu Ala Glu Leu Phe His Met Val Ala Thr 530 535 540 Ala Ala Ala Ile Asp Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly 545 550 555 560 Asn Gly Ile Gly Val Glu Leu Pro Leu Gly Asn Lys Gly Ile Pro Leu 565 570 575 Glu Ile Gly Lys Gly Arg Ile Leu Ile Glu Gly Glu Arg Val Ala Leu 580 585 590 Leu Gly Tyr Gly Ser Ala Val Gln Ser Cys Leu Ala Ala Ala Ser Leu 595 600 605 Leu Glu His His Gly Leu Arg Ala Thr Val Ala Asp Ala Arg Phe Cys 610 615 620 Lys Pro Leu Asp Arg Ser Leu Ile Arg Ser Leu Ala Gln Ser His Glu 625 630 635 640 Val Leu Ile Thr Val Glu Glu Gly Ser Ile Gly Gly Phe Gly Ser His 645 650 655 Val Val Gln Phe Met Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys 660 665 670 Trp Arg Pro Ile Val Leu Pro Asp Cys Tyr Ile Asp His Gly Ser Pro 675 680 685 Val Asp Gln Leu Ser Ala Ala Gly Leu Thr Pro Ser His Ile Ala Ala 690 695 700 Thr Val Phe Asn Leu Leu Gly Gln Thr Arg Glu Ala Leu Glu Val Met 705 710 715 720 Thr 11 2517 DNA Glycine max unsure (2438) n = A, C, G or T 11 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga 60 gctccaccgc ggtggcggcc gctctagaac tagtggatcc cccgggctgc aggaattcgg 120 caccagcatg gatctctccg ctctctcatc ataccgcact ctcgggaagt tacttcctct 180 tccctctcac tctcaatggg gtctccattt cctcgcccac gctcaccgcc tccaccagat 240 gaagaaaagg ccatgtgggg tatatgcatc cctctccgag agtggagagt attattccca 300 ccgaccgcca actcccctac tagacaccgt caactatcct attcatatga agaatctctc 360 tgccaaggag ctgaaacaac tcgcggatga actgcgttct gatgttattt tcagtgtttc 420 tagaactggg ggccatttgg gctcaagcct tggtgtggtg gaactcactg ttgcacttca 480 ctatgtcttc aatgcccctc aggacaagat actgtgggac gttggtcacc agtcttaccc 540 gcataagata ctcaccggta gaagggacca gatgcatacc atgaggcaga caaatggctt 600 atctggcttc accaaacgtt ctgagagtga atttgattgt tttggcactg gtcacagctc 660 caccaccatt tcggcaggac ttggaatggc tgttgggagg gatctgaagg gaagaaagaa 720 taacgtggtt gctgttatag gcgatggtgc catgacagca gggcaagctt atgaagccat 780 gaacaatgct ggatatcttg attctgacat gattgttatt ctaaatgaca acaagcaggt 840 ttctttacca actgctactc ttgatggacc cataccacct gtaggagcct tgagtagcgc 900 tctcagtaga ttacaatcaa ataggcctct tagagaattg agagaggttg ccaagggagt 960 tactaaacga attggaggtc ctatgcatga attggctgca aaagttgacg agtatgctcg 1020 tggcatgatc agtggttctg gatcatcact ttttgaagag cttggactct actatattgg 1080 tcctgttgat ggtcataaca tagatgatct tgttgccatc ctcaacgaag ttaaaagtac 1140 taaaacaacc ggtcctgtat tgattcatgt tatcactgaa aaaggccgtg gataccccta 1200 tgcagaaaag gcagcagaca aataccatgg ggttaccaag tttgacccac caactggaaa 1260 gcaattcaaa tccaaggcta ccactcagtc ttacacaaca tactttgctg aggctttgat 1320 tgcagaagcc gaagctgaca aagacgttgt tgcaatccat gctgctatgg gaggtggaac 1380 tggcatgaat ctcttccatc gccgtttccc aacaagatgc tttgatgtgg ggatagcaga 1440 acagcatgct gttacatttg ctgcaggtct ggcttgtgaa ggtcttaaac ctttctgtgc 1500 aatttactca tcattcatgc agagggctta tgaccaggtg gtgcatgatg tggatttgca 1560 gaagctgcct gtaagatttg caatggacag ggctggatta gttggagcag atggtcccac 1620 acattgtggt tcttttgatg tcacatttat ggcatgcctg cctaacatgg tggtgatggc 1680 tccttctgat gaagccgacc ttttccacat ggttgccacc gcagcagcca ttaatgatcg 1740 acctagttgt tttcgatacc caaggggaaa tggcattggt gttcagctac caactggaaa 1800 taaaggaact cctcttgaga ttgggaaagg taggatattg attgaagggg aaagagtggc 1860 tctcttgggc tatggatcag ctgttcagaa ctgtttggct gcagcttcct tagtggaatg 1920 tcatggcttg cgcttaacag ttgctgatgc acgtttctgc aaaccactgg atcggtccct 1980 gattcgcagc ctggcaaaat cacatgaggt tttaatcaca gttgaagaag gatcaattgg 2040 aggatttggt tctcatgttg ctcagttcat ggcccttgat ggccttctag atggcaaatt 2100 gaagtggcgg ccaatagttc ttccggatcg ttatatcgat catggatcac ctgctgacca 2160 attgtcttta gccggtctta caccatctca catagcagca acagtgttca atgtactagg 2220 acaaacaaga gaggcactag aggtcatgtc atagaaatat taaggggttc aatttttcac 2280 ttcacacgat gtacaaagta taacatgatt caccatgtgt aatatgaaaa agtaatgtaa 2340 tattgtgaaa ttttgaagtg tatgatgtag attgtcatat agtaaaacga ctagttataa 2400 aagagaaaaa tgttaaactt ttctttaaaa aaaaaaanaa naaaaaaaaa actcgagggg 2460 gggcccggta cccaattcgc cctatagtga gtcgtattac gcgcgctaca ctggcgt 2517 12 708 PRT Glycine max 12 Met Asp Leu Ser Ala Leu Ser Ser Tyr Arg Thr Leu Gly Lys Leu Leu 1 5 10 15 Pro Leu Pro Ser His Ser Gln Trp Gly Leu His Phe Leu Ala His Ala 20 25 30 His Arg Leu His Gln Met Lys Lys Arg Pro Cys Gly Val Tyr Ala Ser 35 40 45 Leu Ser Glu Ser Gly Glu Tyr Tyr Ser His Arg Pro Pro Thr Pro Leu 50 55 60 Leu Asp Thr Val Asn Tyr Pro Ile His Met Lys Asn Leu Ser Ala Lys 65 70 75 80 Glu Leu Lys Gln Leu Ala Asp Glu Leu Arg Ser Asp Val Ile Phe Ser 85 90 95 Val Ser Arg Thr Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu 100 105 110 Leu Thr Val Ala Leu His Tyr Val Phe Asn Ala Pro Gln Asp Lys Ile 115 120 125 Leu Trp Asp Val Gly His Gln Ser Tyr Pro His Lys Ile Leu Thr Gly 130 135 140 Arg Arg Asp Gln Met His Thr Met Arg Gln Thr Asn Gly Leu Ser Gly 145 150 155 160 Phe Thr Lys Arg Ser Glu Ser Glu Phe Asp Cys Phe Gly Thr Gly His 165 170 175 Ser Ser Thr Thr Ile Ser Ala Gly Leu Gly Met Ala Val Gly Arg Asp 180 185 190 Leu Lys Gly Arg Lys Asn Asn Val Val Ala Val Ile Gly Asp Gly Ala 195 200 205 Met Thr Ala Gly Gln Ala Tyr Glu Ala Met Asn Asn Ala Gly Tyr Leu 210 215 220 Asp Ser Asp Met Ile Val Ile Leu Asn Asp Asn Lys Gln Val Ser Leu 225 230 235 240 Pro Thr Ala Thr Leu Asp Gly Pro Ile Pro Pro Val Gly Ala Leu Ser 245 250 255 Ser Ala Leu Ser Arg Leu Gln Ser Asn Arg Pro Leu Arg Glu Leu Arg 260 265 270 Glu Val Ala Lys Gly Val Thr Lys Arg Ile Gly Gly Pro Met His Glu 275 280 285 Leu Ala Ala Lys Val Asp Glu Tyr Ala Arg Gly Met Ile Ser Gly Ser 290 295 300 Gly Ser Ser Leu Phe Glu Glu Leu Gly Leu Tyr Tyr Ile Gly Pro Val 305 310 315 320 Asp Gly His Asn Ile Asp Asp Leu Val Ala Ile Leu Asn Glu Val Lys 325 330 335 Ser Thr Lys Thr Thr Gly Pro Val Leu Ile His Val Ile Thr Glu Lys 340 345 350 Gly Arg Gly Tyr Pro Tyr Ala Glu Lys Ala Ala Asp Lys Tyr His Gly 355 360 365 Val Thr Lys Phe Asp Pro Pro Thr Gly Lys Gln Phe Lys Ser Lys Ala 370 375 380 Thr Thr Gln Ser Tyr Thr Thr Tyr Phe Ala Glu Ala Leu Ile Ala Glu 385 390 395 400 Ala Glu Ala Asp Lys Asp Val Val Ala Ile His Ala Ala Met Gly Gly 405 410 415 Gly Thr Gly Met Asn Leu Phe His Arg Arg Phe Pro Thr Arg Cys Phe 420 425 430 Asp Val Gly Ile Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu 435 440 445 Ala Cys Glu Gly Leu Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Met 450 455 460 Gln Arg Ala Tyr Asp Gln Val Val His Asp Val Asp Leu Gln Lys Leu 465 470 475 480 Pro Val Arg Phe Ala Met Asp Arg Ala Gly Leu Val Gly Ala Asp Gly 485 490 495 Pro Thr His Cys Gly Ser Phe Asp Val Thr Phe Met Ala Cys Leu Pro 500 505 510 Asn Met Val Val Met Ala Pro Ser Asp Glu Ala Asp Leu Phe His Met 515 520 525 Val Ala Thr Ala Ala Ala Ile Asn Asp Arg Pro Ser Cys Phe Arg Tyr 530 535 540 Pro Arg Gly Asn Gly Ile Gly Val Gln Leu Pro Thr Gly Asn Lys Gly 545 550 555 560 Thr Pro Leu Glu Ile Gly Lys Gly Arg Ile Leu Ile Glu Gly Glu Arg 565 570 575 Val Ala Leu Leu Gly Tyr Gly Ser Ala Val Gln Asn Cys Leu Ala Ala 580 585 590 Ala Ser Leu Val Glu Cys His Gly Leu Arg Leu Thr Val Ala Asp Ala 595 600 605 Arg Phe Cys Lys Pro Leu Asp Arg Ser Leu Ile Arg Ser Leu Ala Lys 610 615 620 Ser His Glu Val Leu Ile Thr Val Glu Glu Gly Ser Ile Gly Gly Phe 625 630 635 640 Gly Ser His Val Ala Gln Phe Met Ala Leu Asp Gly Leu Leu Asp Gly 645 650 655 Lys Leu Lys Trp Arg Pro Ile Val Leu Pro Asp Arg Tyr Ile Asp His 660 665 670 Gly Ser Pro Ala Asp Gln Leu Ser Leu Ala Gly Leu Thr Pro Ser His 675 680 685 Ile Ala Ala Thr Val Phe Asn Val Leu Gly Gln Thr Arg Glu Ala Leu 690 695 700 Glu Val Met Ser 705 13 526 DNA Triticum aestivum unsure (343) n = A, C, G or T 13 ttgcaatctt gagaaggagg agaggaaaca atggcgctct cgtcgacctt ctccctcccg 60 cggggcttcc tcggcgtgct gcctcaggag caccatttcg ctcccgccgt cgagctccag 120 gccaagccgc tcaagacgcc gaggaggagg tcgtccggca tttctgcgtc gctgtcggag 180 agggaagcag agtaccactc gcagcggccg ccgacgccgc tgctggacac cgtgaactac 240 cccatccaca tgaagaacct gtccctcaag gagctgcagc agctctccga cgaagctgcg 300 ctccgacgtc atcttccact ctccaagaac ggcgggcaac tcnggtcanc ctccgngtcg 360 tcnagctcac gtcncgctng actaactttt caacaaccgc aggacanctc tcnggaantt 420 ggcaacaatc taccgcacaa aatttnacgg ggcggngcat aaatnccaca tgcggagnca 480 aacggacttc cggcttctca ancntccgnc acnatanana gcttct 526 14 106 PRT Triticum aestivum UNSURE (105) Xaa = ANY AMINO ACID 14 Met Ala Leu Ser Ser Thr Phe Ser Leu Pro Arg Gly Phe Leu Gly Val 1 5 10 15 Leu Pro Gln Glu His His Phe Ala Pro Ala Val Glu Leu Gln Ala Lys 20 25 30 Pro Leu Lys Thr Pro Arg Arg Arg Ser Ser Gly Ile Ser Ala Ser Leu 35 40 45 Ser Glu Arg Glu Ala Glu Tyr His Ser Gln Arg Pro Pro Thr Pro Leu 50 55 60 Leu Asp Thr Val Asn Tyr Pro Ile His Met Lys Asn Leu Ser Leu Lys 65 70 75 80 Glu Leu Gln Gln Leu Ser Asp Glu Ala Ala Leu Arg Arg His Leu Pro 85 90 95 Leu Ser Lys Asn Gly Gly Gln Leu Xaa Ser 100 105 15 640 DNA Triticum aestivum unsure (378) n = A, C, G or T 15 ggccttcgac gtggcgttca tggcgtgcct ccccaacatg gtcgtcatgg ccccgtccga 60 cgaggccgag ctgctgaaca tggtcgccac cgccgcggcc atcgacgacc gcccctcgtg 120 cttccgctat ccgaggggca acggcatcgg cgtcccgttg ccggaaaact acaaaggcac 180 tgccatcgag gtcggcaaag gcaggatcat gatcgagggc gagagggtgg cgctgctggg 240 gtacgggtcg gcggtgcagt actgcatggc cgcctcgtcc atcgtggcgc aacacggcct 300 cagggtcacc gtcgccgacg ccaggttctg caagccgttg gaccacgccc tcatcaagag 360 cctcgccaag tccacgangt gatcatcaac gtcnaggaag ctcatcggcg gcttcgctca 420 cacgtggcta attcatggcc tggacggctt ctcaacgnaa actaagtggc ggcggtggtg 480 tcccgacaag tcatcacatg gntaccgcga tanctgtgga ggcggctacc cgtganatgc 540 gcacgtgtaa atctgggaag aaaaaagctc catatacgtc aatncaaaca ttgtgctcan 600 aaaacttnat tgcntaggta aaatatcgta aatattctta 640 16 124 PRT Triticum aestivum 16 Ala Phe Asp Val Ala Phe Met Ala Cys Leu Pro Asn Met Val Val Met 1 5 10 15 Ala Pro Ser Asp Glu Ala Glu Leu Leu Asn Met Val Ala Thr Ala Ala 20 25 30 Ala Ile Asp Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly Asn Gly 35 40 45 Ile Gly Val Pro Leu Pro Glu Asn Tyr Lys Gly Thr Ala Ile Glu Val 50 55 60 Gly Lys Gly Arg Ile Met Ile Glu Gly Glu Arg Val Ala Leu Leu Gly 65 70 75 80 Tyr Gly Ser Ala Val Gln Tyr Cys Met Ala Ala Ser Ser Ile Val Ala 85 90 95 Gln His Gly Leu Arg Val Thr Val Ala Asp Ala Arg Phe Cys Lys Pro 100 105 110 Leu Asp His Ala Leu Ile Lys Ser Leu Ala Lys Ser 115 120 17 1365 DNA Zea mays 17 gagaatgaca agcgcattgt ggtagttcat ggcggcatgg gaatcgatcg atcactccgc 60 ttattccagt ccaggttccc agacagattt tttgacttgg gcatcgctga gcaacatgct 120 gttacctttt ctgctggttt ggcctgcgga ggtctaaagc ctttctgcat aattccatcc 180 acatttcttc agcgagcata tgatcagata attgaagatg tggacatgca aaagatacca 240 gttcgttttg ctatcacaaa tgctggtctg gtaggatctg agggtccaac taattcagga 300 ccatttgata ttacattcat gtcatgcttg ccaaacatga ttgtcatgtc accatctaat 360 gaggatgaac ttattgacat ggtggcaaca gctgcaatga ttgaggacag acctatttgc 420 ttccgctatc ctaggggtgc cattgttggg actagtggaa gtgtaacata tgggaatcca 480 tttgagattg gtaaaggaga gattcttgtc gagggaaaag agatagcttt tcttggctat 540 ggcgaggtgg tccagagatg cttgattgct cgatctcttt tatccaactt tggtattcag 600 gcgacagttg caaacgcgag gttttgcaag ccgcttgaca tcgacctaat cagaacgctg 660 tgtcagcagc atagttttct tatcacagtg gaagaaggaa cggttggtgg ctttggatca 720 cacgtctcac agtttatttc tctcgatggt ctacttgacg gtcgaacaaa ggttcccgtt 780 tctttgtaac tctgcagtgg cgacccattg tgctgccaga caggtacatt gagcatgcat 840 cgctcgcaga gcaacttgac ctggctggcc taactgccca tcacatagct gcaactgcat 900 tgaccctcct agggcgtcat cgtgatgccc ttctgttgat gaagtagggg aagggaccac 960 caagaagaat ggaattggat agataaaagg caatatgtgc agaagttgat tcggaggacg 1020 ctcatcatgc tgttttacga ttgtgttgtc tggatagaac tgaagcgtgc cgtgggaggt 1080 ggccaaatgc acaaatccca aagagggacg acaaagccta tagcaccata gattaatagt 1140 cacggtgtat atactgaaaa gaatttacag accaccgatg taacgttgtt actgtgcatg 1200 ttaatactga aattgtggta agacgccaac tgggagaatg agctagagct gccatgtttc 1260 agttaatgta ataaagctac ttagttttgt atgtaccaat tcattcctta atgttggaat 1320 tcataaccct agcgttcacc tcaaaaaaaa aaaaaaaaaa aaaaa 1365 18 262 PRT Zea mays 18 Glu Asn Asp Lys Arg Ile Val Val Val His Gly Gly Met Gly Ile Asp 1 5 10 15 Arg Ser Leu Arg Leu Phe Gln Ser Arg Phe Pro Asp Arg Phe Phe Asp 20 25 30 Leu Gly Ile Ala Glu Gln His Ala Val Thr Phe Ser Ala Gly Leu Ala 35 40 45 Cys Gly Gly Leu Lys Pro Phe Cys Ile Ile Pro Ser Thr Phe Leu Gln 50 55 60 Arg Ala Tyr Asp Gln Ile Ile Glu Asp Val Asp Met Gln Lys Ile Pro 65 70 75 80 Val Arg Phe Ala Ile Thr Asn Ala Gly Leu Val Gly Ser Glu Gly Pro 85 90 95 Thr Asn Ser Gly Pro Phe Asp Ile Thr Phe Met Ser Cys Leu Pro Asn 100 105 110 Met Ile Val Met Ser Pro Ser Asn Glu Asp Glu Leu Ile Asp Met Val 115 120 125 Ala Thr Ala Ala Met Ile Glu Asp Arg Pro Ile Cys Phe Arg Tyr Pro 130 135 140 Arg Gly Ala Ile Val Gly Thr Ser Gly Ser Val Thr Tyr Gly Asn Pro 145 150 155 160 Phe Glu Ile Gly Lys Gly Glu Ile Leu Val Glu Gly Lys Glu Ile Ala 165 170 175 Phe Leu Gly Tyr Gly Glu Val Val Gln Arg Cys Leu Ile Ala Arg Ser 180 185 190 Leu Leu Ser Asn Phe Gly Ile Gln Ala Thr Val Ala Asn Ala Arg Phe 195 200 205 Cys Lys Pro Leu Asp Ile Asp Leu Ile Arg Thr Leu Cys Gln Gln His 210 215 220 Ser Phe Leu Ile Thr Val Glu Glu Gly Thr Val Gly Gly Phe Gly Ser 225 230 235 240 His Val Ser Gln Phe Ile Ser Leu Asp Gly Leu Leu Asp Gly Arg Thr 245 250 255 Lys Val Pro Val Ser Leu 260 19 1488 DNA Zea mays 19 ggcacgagag cagatcggtg gctcagtgca cgagctggcg gcgaaggtgg acgagtacgc 60 ccgcggcatg atcagcgggc ccggctcctc gctcttcgag gagctcggtc tctactacat 120 cggccccgtc gacggccaca acatcgacga cctcatcacc atcctcaacg acgtcaagag 180 caccaagacc accggccccg tcctcatcca cgtcgtcacc gagaagggcc gcggctaccc 240 ctacgccgag cgagccgccg acaagtacca cggtgtcgcc aagtttgatc cggcgaccgg 300 gaagcagttc aagtcccccg ccaagacgct gtcctacacc aactacttcg ccgaggcgct 360 catcgccgag gcggagcagg acagcaagat cgtggccatc cacgcggcca tgggcggcgg 420 cacggggctc aactacttcc tccgccgctt cccgagccgg tgcttcgacg tcgggatcgc 480 ggagcagcac gccgtcacgt tcgcggccgg cctggcctgc gagggcctca agcccttctg 540 cgccatctac tcgtctttcc tgcagcgcgg ctacgaccag gtcgtgcacg acgtcgatct 600 gcagaagcta ccggtgcggt tcgccatgga cagggccggg ctggtcggcg cggacgggcc 660 gacccactgc ggtgcgttcg acgtcgcgta catggcctgc ctgcccaaca tggtcgtcat 720 ggccccgtcc gacgaggccg agctctgcca catggtcgcc accgccgcgg ccatcgacga 780 ccgcccgtcc tgcttccgct acccgagagg caacggcgtt ggcgtcccgt tgccgcccaa 840 ctacaaaggc actcccctcg aggtcggcaa aggcaggatc ctgcttgagg gcgaccgggt 900 ggcgctgctg gggtacgggt cggcagtgca gtactgcctg actgccgcgt ccctggtgca 960 gcgccacggc ctcaaggtca ccgtcgccga cgcgaggttc tgcaagccgc tggaccacgc 1020 cctgatcagg agcctggcca agtcccacga ggtgctcatc accgtggagg aaggctccat 1080 cggcgggttc ggctcgcacg tcgcccagtt catggccctg gacggccttc tcgacggcaa 1140 actcaagtgg cgaccgctgg tgcttcctga caggtacatc gaccatggat cgccggccga 1200 tcagctggcc gaggctgggc tgacgccgtc acacatcgcc gcgtcggtgt tcaacatcct 1260 ggggcagaac agggaggctc ttgccatcat ggcagtgcca aacgcgtaga acttgtgctg 1320 atctgggcct atagagatga ttgtacattt tgtcgttaac tagagtgtct gaacttggga 1380 gattagtctt ctttggatga aagtgtcgcc ggaacaacag ttaccgtttc tttttttgaa 1440 agagaaaggc aaaagatttg ccattccaat aaaaaaaaaa aaaaaaaa 1488 20 435 PRT Zea mays 20 Ala Arg Glu Gln Ile Gly Gly Ser Val His Glu Leu Ala Ala Lys Val 1 5 10 15 Asp Glu Tyr Ala Arg Gly Met Ile Ser Gly Pro Gly Ser Ser Leu Phe 20 25 30 Glu Glu Leu Gly Leu Tyr Tyr Ile Gly Pro Val Asp Gly His Asn Ile 35 40 45 Asp Asp Leu Ile Thr Ile Leu Asn Asp Val Lys Ser Thr Lys Thr Thr 50 55 60 Gly Pro Val Leu Ile His Val Val Thr Glu Lys Gly Arg Gly Tyr Pro 65 70 75 80 Tyr Ala Glu Arg Ala Ala Asp Lys Tyr His Gly Val Ala Lys Phe Asp 85 90 95 Pro Ala Thr Gly Lys Gln Phe Lys Ser Pro Ala Lys Thr Leu Ser Tyr 100 105 110 Thr Asn Tyr Phe Ala Glu Ala Leu Ile Ala Glu Ala Glu Gln Asp Ser 115 120 125 Lys Ile Val Ala Ile His Ala Ala Met Gly Gly Gly Thr Gly Leu Asn 130 135 140 Tyr Phe Leu Arg Arg Phe Pro Ser Arg Cys Phe Asp Val Gly Ile Ala 145 150 155 160 Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Cys Glu Gly Leu 165 170 175 Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Leu Gln Arg Gly Tyr Asp 180 185 190 Gln Val Val His Asp Val Asp Leu Gln Lys Leu Pro Val Arg Phe Ala 195 200 205 Met Asp Arg Ala Gly Leu Val Gly Ala Asp Gly Pro Thr His Cys Gly 210 215 220 Ala Phe Asp Val Ala Tyr Met Ala Cys Leu Pro Asn Met Val Val Met 225 230 235 240 Ala Pro Ser Asp Glu Ala Glu Leu Cys His Met Val Ala Thr Ala Ala 245 250 255 Ala Ile Asp Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly Asn Gly 260 265 270 Val Gly Val Pro Leu Pro Pro Asn Tyr Lys Gly Thr Pro Leu Glu Val 275 280 285 Gly Lys Gly Arg Ile Leu Leu Glu Gly Asp Arg Val Ala Leu Leu Gly 290 295 300 Tyr Gly Ser Ala Val Gln Tyr Cys Leu Thr Ala Ala Ser Leu Val Gln 305 310 315 320 Arg His Gly Leu Lys Val Thr Val Ala Asp Ala Arg Phe Cys Lys Pro 325 330 335 Leu Asp His Ala Leu Ile Arg Ser Leu Ala Lys Ser His Glu Val Leu 340 345 350 Ile Thr Val Glu Glu Gly Ser Ile Gly Gly Phe Gly Ser His Val Ala 355 360 365 Gln Phe Met Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys Trp Arg 370 375 380 Pro Leu Val Leu Pro Asp Arg Tyr Ile Asp His Gly Ser Pro Ala Asp 385 390 395 400 Gln Leu Ala Glu Ala Gly Leu Thr Pro Ser His Ile Ala Ala Ser Val 405 410 415 Phe Asn Ile Leu Gly Gln Asn Arg Glu Ala Leu Ala Ile Met Ala Val 420 425 430 Pro Asn Ala 435 21 461 DNA Zea mays 21 gagccggcgg cggcggccac gtcgtcggga ccgtggaaga tcgacttctc cggcgagaag 60 ccgccgacgc cgctgctgga caccgtgaac tacccgctcc acatgaagaa cctgtcgatc 120 ttggagctgg agcagctggc ggcggagctc cgcgcggagg tcgtgcacac cgtgtccaag 180 accggcgggc acctgagctc cagcctgggc gttgtggagc tgtcggtggc gctgcaccac 240 gtgttcgaca ccccggagga caagatcatc tgggacgtgg gccaccaggc gtacccgcac 300 aagatcctga cggggcggcg gtcgcggatg cacaccatcc gccagacctc cgggctggcg 360 gggttcccca agcgcgacga gagcgcgcac gacgcgttcg gggtcggcca cagctccaac 420 agcatctcgg cggcgctggg catggccgtt gcgcgggacc t 461 22 153 PRT Zea mays 22 Glu Pro Ala Ala Ala Ala Thr Ser Ser Gly Pro Trp Lys Ile Asp Phe 1 5 10 15 Ser Gly Glu Lys Pro Pro Thr Pro Leu Leu Asp Thr Val Asn Tyr Pro 20 25 30 Leu His Met Lys Asn Leu Ser Ile Leu Glu Leu Glu Gln Leu Ala Ala 35 40 45 Glu Leu Arg Ala Glu Val Val His Thr Val Ser Lys Thr Gly Gly His 50 55 60 Leu Ser Ser Ser Leu Gly Val Val Glu Leu Ser Val Ala Leu His His 65 70 75 80 Val Phe Asp Thr Pro Glu Asp Lys Ile Ile Trp Asp Val Gly His Gln 85 90 95 Ala Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Ser Arg Met His Thr 100 105 110 Ile Arg Gln Thr Ser Gly Leu Ala Gly Phe Pro Lys Arg Asp Glu Ser 115 120 125 Ala His Asp Ala Phe Gly Val Gly His Ser Ser Asn Ser Ile Ser Ala 130 135 140 Ala Leu Gly Met Ala Val Ala Arg Asp 145 150 23 698 DNA Zea mays unsure (418) n = A, C, G or T 23 gagcgcgacg ccctgccagt agccgccgca accggccgcc ggtcccgcgc gaggggagga 60 ggagatcact gcggcggcgt cttctgccgg tttgaggatt cggggcatca ctgcagcagc 120 tggcgccagg ctccagcatg gacacggcgt ttctgagtcc tccgcttgcc cgtaatctgg 180 tttatgacga gtttgccgtt cttcacccca ctagctaccc ttttcatact cttcggtatt 240 tgagatgcaa tccaatgtat tcgagaccgc tgctaacaat agcaccagcc tcaccatcaa 300 ggggcttgat tcagagagtg gccgcactac ctgatgttga tgatttcttc tgggagaagg 360 atcctactcc aatacttgac acaattgatg cacccattca tttgaaaaat ctatctanag 420 ctcaaagcag ttagcccgat gaagtttgtt canaaaatag ctttcataat tgtcanaaaa 480 tgccaacccg tgtggtgctg atcgctcant tgtggagctg acaattgcta tacattatgt 540 gttcaatgcc ccaatggata agaaactatg ggatgctggn caaacatgca tatgcnnaca 600 agattcttac aaggaaggcg ctcttctctt ccattctatt acacagaaaa aatggccttt 660 ctnggtttaa cntnncgttt ttgataaccg antatgat 698 24 35 PRT Zea mays 24 Gln Arg Val Ala Ala Leu Pro Asp Val Asp Asp Phe Phe Trp Glu Lys 1 5 10 15 Asp Pro Thr Pro Ile Leu Asp Thr Ile Asp Ala Pro Ile His Leu Lys 20 25 30 Asn Leu Ser 35 25 2618 DNA Oryza sativa 25 gcacgagctt acatgtcctt tctccacctc ggtggtcatc agctagacag ctatcgcgcg 60 ccgtcccacc accatcttgc tccactacgc ggaccaccgc gcgcgagcag agcatctcct 120 cactctctag cttgctccag tttcgcgtag ctgcgtgaca gttcaattga actctctgga 180 ttcgttggtt acttcgtctg agctgctgca gcgttgagga ggaggaggag caatggcgct 240 cacgacgttc tccatttcga gaggaggctt cgtcggcgcg ctgccgcagg aggggcattt 300 cgctccggcg gcggcggagc tcagtctcca caagctccag agcaggccac acaaggctag 360 gcggaggtcg tcgtcgagca tctcggcgtc gctgtccacg gagagggagg cggcggagta 420 ccactcgcag cggccaccga cgccgctgct ggacacggtc aactacccca tccacatgaa 480 gaacctgtcc ctcaaggagc tccagcagct cgccgacgag ctccgctccg acgtcatctt 540 ccacgtctcc aagaccgggg gacatctcgg gtccagcctc ggcgtcgtcg agctcaccgt 600 cgcgctccac tacgtgttca acacgcctca ggacaagatc ctctgggacg tcggccacca 660 gtcgtaccct cacaagattc tgaccgggcg gcgcgacaag atgccgacga tgcgtcagac 720 caacggcttg tcgggattca ccaagcggtc ggagagcgag tacgactcct tcggcaccgg 780 ccacagctcc accaccatct ccgccgccct cgggatggcg gtggggaggg atctcaaggg 840 agggaagaac aacgtggtgg cggtgatcgg cgacggcgcc atgacggccg ggcaggcgta 900 cgaggcgatg aataacgcgg ggtatctcga ctccgatatg atcgtgattc tcaacgacaa 960 caagcaggtg tcgctgccga cggcgacgct cgacgggccg gcgccgccgg tgggcgcgct 1020 cagcagcgcc ctcagcaagc tgcagtccag ccgcccactc agggagctca gggaggtggc 1080 aaagggcgtg acgaagcaaa tcggagggtc ggtgcacgag ctggcggcga aggtggacga 1140 gtacgcccgc ggcatgatca gcggctccgg ctcgacgctc ttcgaggagc tcggcctcta 1200 ctacatcggc cccgtcgacg gccacaacat cgacgacctc atcaccatcc tccgcgaggt 1260 caagagcacc aagaccacag gcccggtgct catccacgtc gtcaccgaga aaggccgcgg 1320 ctacccctac gccgagcgcg ccgccgacaa gtaccacggc gtggcgaagt tcgatccggc 1380 gacggggaag cagttcaagt cgccggcgaa gacgctgtcg tacacgaact acttcgcgga 1440 ggcgctcatc gccgaggcgg agcaggacaa cagggtcgtg gccatccacg cggccatggg 1500 gggaggcacg gggctcaact acttcctccg ccgcttcccg aaccggtgct tcgacgtcgg 1560 gatcgccgag cagcacgccg tcacgttcgc cgccggcctc gcctgcgagg gcctcaagcc 1620 gttctgcgcc atctactcct ccttcctgca gagaggctac gaccaggtgg tgcacgacgt 1680 ggacctccag aagctgccgg tgaggttcgc catggacagg gccgggctcg tgggcgccga 1740 cgggccgacg cactgcggcg cgttcgacgt cacctacatg gcgtgcctgc cgaacatggt 1800 cgtcatggcc ccgtccgacg aggcggagct ctgccacatg gtcgccaccg ccgcggccat 1860 cgacgaccgc ccctcctgct tccgctaccc aagaggcaac ggcatcggcg tcccgctacc 1920 acccaactac aaaggcgttc ccctcgaggt aggcaaaggg agggtactgc tggagggcga 1980 gagggtggcg ctgcttgggt acggttcggc ggtgcagtac tgcctcgccg cagcgtcgct 2040 ggtggagcgg cacggcctca aggtgaccgt cgccgacgcg aggttctgca agccgctgga 2100 ccaaacgctc atcaggaggc tggccagctc ccacgaggtg ctcctcaccg tcgaggaagg 2160 ctccatcggc gggttcggct cccacgtcgc gcagttcatg gccctcgacg gcctcctcga 2220 cggcaaactc aagtggcggc cgctggtgct acccgacagg tacatcgacc acgggtcacc 2280 ggcggatcag ctggcggagg cagggctgac gccgtcgcac atcgcggcga cggtgttcaa 2340 cgtgctgggc caggcgaggg aggcgctcgc catcatgacg gtgcccaacg cgtagcagat 2400 gcgtggcgcc tctggtagag acaatgcttt gtacatgtag agatcagtga attgtatatt 2460 agtcggcgtc gggataaata ttgattagtg atgctgaggg gaacagttac agtttttttg 2520 ctcttcagtt gttcgtggac ggagacccgg ctgctcgatg ttcgatcgct tgtatatcta 2580 agaaatgttg taagtggata aaaaaaaaaa aaaaaaaa 2618 26 720 PRT Oryza sativa 26 Met Ala Leu Thr Thr Phe Ser Ile Ser Arg Gly Gly Phe Val Gly Ala 1 5 10 15 Leu Pro Gln Glu Gly His Phe Ala Pro Ala Ala Ala Glu Leu Ser Leu 20 25 30 His Lys Leu Gln Ser Arg Pro His Lys Ala Arg Arg Arg Ser Ser Ser 35 40 45 Ser Ile Ser Ala Ser Leu Ser Thr Glu Arg Glu Ala Ala Glu Tyr His 50 55 60 Ser Gln Arg Pro Pro Thr Pro Leu Leu Asp Thr Val Asn Tyr Pro Ile 65 70 75 80 His Met Lys Asn Leu Ser Leu Lys Glu Leu Gln Gln Leu Ala Asp Glu 85 90 95 Leu Arg Ser Asp Val Ile Phe His Val Ser Lys Thr Gly Gly His Leu 100 105 110 Gly Ser Ser Leu Gly Val Val Glu Leu Thr Val Ala Leu His Tyr Val 115 120 125 Phe Asn Thr Pro Gln Asp Lys Ile Leu Trp Asp Val Gly His Gln Ser 130 135 140 Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Lys Met Pro Thr Met 145 150 155 160 Arg Gln Thr Asn Gly Leu Ser Gly Phe Thr Lys Arg Ser Glu Ser Glu 165 170 175 Tyr Asp Ser Phe Gly Thr Gly His Ser Ser Thr Thr Ile Ser Ala Ala 180 185 190 Leu Gly Met Ala Val Gly Arg Asp Leu Lys Gly Gly Lys Asn Asn Val 195 200 205 Val Ala Val Ile Gly Asp Gly Ala Met Thr Ala Gly Gln Ala Tyr Glu 210 215 220 Ala Met Asn Asn Ala Gly Tyr Leu Asp Ser Asp Met Ile Val Ile Leu 225 230 235 240 Asn Asp Asn Lys Gln Val Ser Leu Pro Thr Ala Thr Leu Asp Gly Pro 245 250 255 Ala Pro Pro Val Gly Ala Leu Ser Ser Ala Leu Ser Lys Leu Gln Ser 260 265 270 Ser Arg Pro Leu Arg Glu Leu Arg Glu Val Ala Lys Gly Val Thr Lys 275 280 285 Gln Ile Gly Gly Ser Val His Glu Leu Ala Ala Lys Val Asp Glu Tyr 290 295 300 Ala Arg Gly Met Ile Ser Gly Ser Gly Ser Thr Leu Phe Glu Glu Leu 305 310 315 320 Gly Leu Tyr Tyr Ile Gly Pro Val Asp Gly His Asn Ile Asp Asp Leu 325 330 335 Ile Thr Ile Leu Arg Glu Val Lys Ser Thr Lys Thr Thr Gly Pro Val 340 345 350 Leu Ile His Val Val Thr Glu Lys Gly Arg Gly Tyr Pro Tyr Ala Glu 355 360 365 Arg Ala Ala Asp Lys Tyr His Gly Val Ala Lys Phe Asp Pro Ala Thr 370 375 380 Gly Lys Gln Phe Lys Ser Pro Ala Lys Thr Leu Ser Tyr Thr Asn Tyr 385 390 395 400 Phe Ala Glu Ala Leu Ile Ala Glu Ala Glu Gln Asp Asn Arg Val Val 405 410 415 Ala Ile His Ala Ala Met Gly Gly Gly Thr Gly Leu Asn Tyr Phe Leu 420 425 430 Arg Arg Phe Pro Asn Arg Cys Phe Asp Val Gly Ile Ala Glu Gln His 435 440 445 Ala Val Thr Phe Ala Ala Gly Leu Ala Cys Glu Gly Leu Lys Pro Phe 450 455 460 Cys Ala Ile Tyr Ser Ser Phe Leu Gln Arg Gly Tyr Asp Gln Val Val 465 470 475 480 His Asp Val Asp Leu Gln Lys Leu Pro Val Arg Phe Ala Met Asp Arg 485 490 495 Ala Gly Leu Val Gly Ala Asp Gly Pro Thr His Cys Gly Ala Phe Asp 500 505 510 Val Thr Tyr Met Ala Cys Leu Pro Asn Met Val Val Met Ala Pro Ser 515 520 525 Asp Glu Ala Glu Leu Cys His Met Val Ala Thr Ala Ala Ala Ile Asp 530 535 540 Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly Asn Gly Ile Gly Val 545 550 555 560 Pro Leu Pro Pro Asn Tyr Lys Gly Val Pro Leu Glu Val Gly Lys Gly 565 570 575 Arg Val Leu Leu Glu Gly Glu Arg Val Ala Leu Leu Gly Tyr Gly Ser 580 585 590 Ala Val Gln Tyr Cys Leu Ala Ala Ala Ser Leu Val Glu Arg His Gly 595 600 605 Leu Lys Val Thr Val Ala Asp Ala Arg Phe Cys Lys Pro Leu Asp Gln 610 615 620 Thr Leu Ile Arg Arg Leu Ala Ser Ser His Glu Val Leu Leu Thr Val 625 630 635 640 Glu Glu Gly Ser Ile Gly Gly Phe Gly Ser His Val Ala Gln Phe Met 645 650 655 Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys Trp Arg Pro Leu Val 660 665 670 Leu Pro Asp Arg Tyr Ile Asp His Gly Ser Pro Ala Asp Gln Leu Ala 675 680 685 Glu Ala Gly Leu Thr Pro Ser His Ile Ala Ala Thr Val Phe Asn Val 690 695 700 Leu Gly Gln Ala Arg Glu Ala Leu Ala Ile Met Thr Val Pro Asn Ala 705 710 715 720 27 1991 DNA Oryza sativa 27 gcacgaggct ggtcagcata catatgcaca caagattctc acaggaaggc gctcactctt 60 tcatactatt aagcaaagaa aggggctttc aggtttcaca tcccgtttcg agagcgaata 120 tgatcccttt ggtgcaggac atggatgcaa tagtctctcc gcaggccttg ggatggcagt 180 cgcaagggat ctaggtggga ggaaaaaccg aatagtaaca gttataagta actggacaac 240 tatggctggt caggtgtatg aggcaatggg tcatgccggt ttccttgatt ctaacatggt 300 agtgatttta aatgacagcc ggcacacctt gcttcctaaa gcagatagcc aatcaaagat 360 gtctattaat gccctctcta gtgctctgag caaggttcaa tccagcaaag gatttagaaa 420 gtttagggag gctgcaaagg gactttccaa atggtttggt aaagggatgc atgaatttgc 480 tgccaaaatt gatgagtatg cccgtggtat gataggtcct catggagcaa ctctttttga 540 agaacttgga taatattata ttgggcctat tgatgggaat aacattgatg atctcatttg 600 tgtactcaag gaggtttcta ctctagattc taccggccca gtacttgtgc atgtaatcac 660 tgagaatgaa aaagactcag gtggagaatt taatagtgag attactcccg acgaggaagg 720 gcctccagac tcaagccaag acattctaaa gtttttagaa aatggtcttt ctaggacata 780 taatgattgc tttgtagaat cactaatagc agaagcagag aatgacaagc atattgtggt 840 ggttcatgga ggcatgggaa tagatcgatc aatccaatta tttcagtcca gatttccgga 900 cagatttttc gatttgggta tcgccgagca acatgctgtt acgttttctg ctggtttggc 960 atgcggaggc ttaaagcctt tctgcataat tccatccacc tttctccagc gagcatatga 1020 tcagatagtc gaagatgtgg acatgcaaaa gataccagtt cgctttgcaa tcacaagtgc 1080 aggtctggtg ggatctgaag gcccgactaa ctcaggacca tttgatatta cattcatgtc 1140 atgcctgcca aacatgatcg tcatgtcacc atctaatgag gatgaactta ttgacatggt 1200 ggcaacagct gcaatggttg aggacagacc catttgcttc cggtatccca agggtgccat 1260 cgttgggact agtggcactt tagcatatgg gaatccactt gagattggta aaggagagat 1320 tcttgctgag gggaaagaga tagcttttct tggttatggt gatgtggtcc agagatgctt 1380 gatagctcga tctcttctgt tcaactttgg catccaggca actgttgcta atgcgagatt 1440 ttgcaagcca cttgacattg atctgataag aatgttgtgc cagcaacacg atttcctaat 1500 caccgtggaa gaaggaacgg ttggtggttt tggctcacac gtctcgcaat ttatttcact 1560 cgatgggttg cttgatggca aaataaagtg gcgacccatt gtactaccag acaggtacat 1620 cgaacacgct tcgctcacag agcagctcga catggctggg ttgactgctc atcacatcgc 1680 agcaaccgca ctgacccttt tagggcgaca ccgagacgca cttttgttga tgaagtaaga 1740 aggaaaaatg agctagaaaa gaatgaaaag ttgtgcagca agtttgagct ggtagaagac 1800 agccaaattg ctgtttcatg gatattcttc agtctttcag aggaaactga gattgccatg 1860 gcagatacag cctgtgtgca ccactgaaag agcttgcaag tttttatctg tgctccagat 1920 gcttactgta atctgttcat gggggctgta catactataa accctgtttt gatgatgatt 1980 atgttaatgt t 1991 28 578 PRT Oryza sativa UNSURE (184) Xaa = ANY AMINO ACID 28 His Glu Ala Gly Gln His Thr Tyr Ala His Lys Ile Leu Thr Gly Arg 1 5 10 15 Arg Ser Leu Phe His Thr Ile Lys Gln Arg Lys Gly Leu Ser Gly Phe 20 25 30 Thr Ser Arg Phe Glu Ser Glu Tyr Asp Pro Phe Gly Ala Gly His Gly 35 40 45 Cys Asn Ser Leu Ser Ala Gly Leu Gly Met Ala Val Ala Arg Asp Leu 50 55 60 Gly Gly Arg Lys Asn Arg Ile Val Thr Val Ile Ser Asn Trp Thr Thr 65 70 75 80 Met Ala Gly Gln Val Tyr Glu Ala Met Gly His Ala Gly Phe Leu Asp 85 90 95 Ser Asn Met Val Val Ile Leu Asn Asp Ser Arg His Thr Leu Leu Pro 100 105 110 Lys Ala Asp Ser Gln Ser Lys Met Ser Ile Asn Ala Leu Ser Ser Ala 115 120 125 Leu Ser Lys Val Gln Ser Ser Lys Gly Phe Arg Lys Phe Arg Glu Ala 130 135 140 Ala Lys Gly Leu Ser Lys Trp Phe Gly Lys Gly Met His Glu Phe Ala 145 150 155 160 Ala Lys Ile Asp Glu Tyr Ala Arg Gly Met Ile Gly Pro His Gly Ala 165 170 175 Thr Leu Phe Glu Glu Leu Gly Xaa Tyr Tyr Ile Gly Pro Ile Asp Gly 180 185 190 Asn Asn Ile Asp Asp Leu Ile Cys Val Leu Lys Glu Val Ser Thr Leu 195 200 205 Asp Ser Thr Gly Pro Val Leu Val His Val Ile Thr Glu Asn Glu Lys 210 215 220 Asp Ser Gly Gly Glu Phe Asn Ser Glu Ile Thr Pro Asp Glu Glu Gly 225 230 235 240 Pro Pro Asp Ser Ser Gln Asp Ile Leu Lys Phe Leu Glu Asn Gly Leu 245 250 255 Ser Arg Thr Tyr Asn Asp Cys Phe Val Glu Ser Leu Ile Ala Glu Ala 260 265 270 Glu Asn Asp Lys His Ile Val Val Val His Gly Gly Met Gly Ile Asp 275 280 285 Arg Ser Ile Gln Leu Phe Gln Ser Arg Phe Pro Asp Arg Phe Phe Asp 290 295 300 Leu Gly Ile Ala Glu Gln His Ala Val Thr Phe Ser Ala Gly Leu Ala 305 310 315 320 Cys Gly Gly Leu Lys Pro Phe Cys Ile Ile Pro Ser Thr Phe Leu Gln 325 330 335 Arg Ala Tyr Asp Gln Ile Val Glu Asp Val Asp Met Gln Lys Ile Pro 340 345 350 Val Arg Phe Ala Ile Thr Ser Ala Gly Leu Val Gly Ser Glu Gly Pro 355 360 365 Thr Asn Ser Gly Pro Phe Asp Ile Thr Phe Met Ser Cys Leu Pro Asn 370 375 380 Met Ile Val Met Ser Pro Ser Asn Glu Asp Glu Leu Ile Asp Met Val 385 390 395 400 Ala Thr Ala Ala Met Val Glu Asp Arg Pro Ile Cys Phe Arg Tyr Pro 405 410 415 Lys Gly Ala Ile Val Gly Thr Ser Gly Thr Leu Ala Tyr Gly Asn Pro 420 425 430 Leu Glu Ile Gly Lys Gly Glu Ile Leu Ala Glu Gly Lys Glu Ile Ala 435 440 445 Phe Leu Gly Tyr Gly Asp Val Val Gln Arg Cys Leu Ile Ala Arg Ser 450 455 460 Leu Leu Phe Asn Phe Gly Ile Gln Ala Thr Val Ala Asn Ala Arg Phe 465 470 475 480 Cys Lys Pro Leu Asp Ile Asp Leu Ile Arg Met Leu Cys Gln Gln His 485 490 495 Asp Phe Leu Ile Thr Val Glu Glu Gly Thr Val Gly Gly Phe Gly Ser 500 505 510 His Val Ser Gln Phe Ile Ser Leu Asp Gly Leu Leu Asp Gly Lys Ile 515 520 525 Lys Trp Arg Pro Ile Val Leu Pro Asp Arg Tyr Ile Glu His Ala Ser 530 535 540 Leu Thr Glu Gln Leu Asp Met Ala Gly Leu Thr Ala His His Ile Ala 545 550 555 560 Ala Thr Ala Leu Thr Leu Leu Gly Arg His Arg Asp Ala Leu Leu Leu 565 570 575 Met Lys 29 898 DNA Triticum aestivum unsure (145) n = A, C, G or T 29 ccttagagtg ggcttcaatg ggtcctaccc aaacatggta gttatgcccc ctccggacga 60 ggccgagatg ctaaacatgg tggcaaccgc ggcggccatc gacgaccgcc cctcgtgctt 120 ccgctatccg aggggcaacg gcatnggcgt cccgttgccg gaaaactaca aaggcaccgc 180 catcgaggtc ggcaaaggca ggatcataat cgagggcgag agggtggcgc tgctggggta 240 cgggtcggcg gtgcagtact gcatggccgc ctcgtccatc gtggcgcacc acggcctcag 300 ggtcaccgtc gccgacgcca ggttctgcaa gccgttggac cacgccctca tcaggagcct 360 cgccaagtcc cacgaggtga tcatcaccgt cgaggaaggc tccatcggcg gcttcggttc 420 acacgtggct cagttcatgg ccctggatgg ccttctggac ggcaaactta agtggcggcc 480 ggtggtgctt cccgacaagt acatcgacca tggatcaccg gccgatcagc tggtggaagc 540 cgggctgacg ccgtcgcaca tcgccgcgac ggtgttcaac atcctggggc aggcaagaga 600 ggccctcgcc atcatgacgg tgcagaatgc ctagagccag tgtgctgcct cctatagaga 660 accttgtaca ttttggtcgt taggtgattc agagagatta gtcggcgtca gaaaattaaa 720 tgatcctcat caagggaaac gttggtagtt tttcgttctt tggtgcactg acgttgatgt 780 acatggttaa ttgttcgtgg agtggacaca tacgttgtct ttgtatctgt gaaatgtgta 840 cgtatgttta ttggaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 898 30 210 PRT Triticum aestivum UNSURE (48) Xaa = ANY AMINO ACID 30 Leu Arg Val Gly Phe Asn Gly Ser Tyr Pro Asn Met Val Val Met Pro 1 5 10 15 Pro Pro Asp Glu Ala Glu Met Leu Asn Met Val Ala Thr Ala Ala Ala 20 25 30 Ile Asp Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly Asn Gly Xaa 35 40 45 Gly Val Pro Leu Pro Glu Asn Tyr Lys Gly Thr Ala Ile Glu Val Gly 50 55 60 Lys Gly Arg Ile Ile Ile Glu Gly Glu Arg Val Ala Leu Leu Gly Tyr 65 70 75 80 Gly Ser Ala Val Gln Tyr Cys Met Ala Ala Ser Ser Ile Val Ala His 85 90 95 His Gly Leu Arg Val Thr Val Ala Asp Ala Arg Phe Cys Lys Pro Leu 100 105 110 Asp His Ala Leu Ile Arg Ser Leu Ala Lys Ser His Glu Val Ile Ile 115 120 125 Thr Val Glu Glu Gly Ser Ile Gly Gly Phe Gly Ser His Val Ala Gln 130 135 140 Phe Met Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys Trp Arg Pro 145 150 155 160 Val Val Leu Pro Asp Lys Tyr Ile Asp His Gly Ser Pro Ala Asp Gln 165 170 175 Leu Val Glu Ala Gly Leu Thr Pro Ser His Ile Ala Ala Thr Val Phe 180 185 190 Asn Ile Leu Gly Gln Ala Arg Glu Ala Leu Ala Ile Met Thr Val Gln 195 200 205 Asn Ala 210 31 1404 DNA Triticum aestivum 31 ttgcaatctt gagaaggagg agaggaaaca atggcgctct cgtcgacctt ctccctcccg 60 cggggcttcc tcggcgtgct gcctcaggag caccatttcg ctcccgccgt cgagctccag 120 gccaagccgc tcaagacgcc gaggaggagg tcgtccggca tttctgcgtc gctgtcggag 180 agggaagcag agtaccactc gcagcggccg ccgacgccgc tgctggacac cgtgaactac 240 cccatccaca tgaagaacct gtccctcaag gagctgcagc agctctccga cgagctgcgc 300 tccgacgtca tcttccacgt ctccaagacc ggcggccacc tcgggtccag cctcggcgtc 360 gtcgagctca ccgtcgcgct gcactacgtc ttcaacaccc cgcaggacaa gctcctctgg 420 gacgtcggcc accagtcgta cccgcacaag attctgacgg ggcggcgcga taagatgccg 480 acgatgcggc agaccaacgg cctgtccggc ttcgtcaagc gctccgagag cgagtacgac 540 agcttcggca ccggccacag ctccaccacc atctccgccg ccctcgggat ggccgtcggg 600 agggacctca agggcgcgaa gaacaacgtg gtggcggtga ttggggacgg ggccatgacg 660 gccgggcagg cgtacgaggc gatgaacaac gccggctacc tcgactcgga catgatcgtg 720 atcctcaacg acaacaagca ggtgtcgctg ccgacggcga cgctcgacgg gccggcgccg 780 cccgtgggcg cgctcagcgg cgccctcagc aagctgcagt ccagccggcc gctcagggag 840 ctgagggagg tggccaaggg agtgacgaag caaatcggcg ggtcggtgca cgagatcgcg 900 gccaaggtgg acgagtacgc ccgcggcatg atcagcggct ccgggtcgtc gctcttcgag 960 gagctcgggc tgtattacat cggccccgtc gacggccaca acattgacga cctcatcacc 1020 atccttcggg aggtcaaggg caccaagacc accgggccgg tgctcatcca tgtcatcacc 1080 gagaaaggcc gcggctaccc ctacgccgag cgagcctccg acaagtacca acgggtggca 1140 aagttcgatc cggcgaccgg gaggcagttc aagggtccgg ccaagacgcc ttcctacaac 1200 aactacttcg cggagccgct catagcccag gcggggcaag acagcaagat cgtggcattc 1260 cacccggcca tggggggcgg gacggggctc aactacttcc tccgccgctt ccccaaccgg 1320 ggcttccaag tcgaatccgc taaacagaac gccgtaaccc ttcccggccg cctggccggc 1380 aagggggtta aacccttctg cgca 1404 32 458 PRT Triticum aestivum 32 Met Ala Leu Ser Ser Thr Phe Ser Leu Pro Arg Gly Phe Leu Gly Val 1 5 10 15 Leu Pro Gln Glu His His Phe Ala Pro Ala Val Glu Leu Gln Ala Lys 20 25 30 Pro Leu Lys Thr Pro Arg Arg Arg Ser Ser Gly Ile Ser Ala Ser Leu 35 40 45 Ser Glu Arg Glu Ala Glu Tyr His Ser Gln Arg Pro Pro Thr Pro Leu 50 55 60 Leu Asp Thr Val Asn Tyr Pro Ile His Met Lys Asn Leu Ser Leu Lys 65 70 75 80 Glu Leu Gln Gln Leu Ser Asp Glu Leu Arg Ser Asp Val Ile Phe His 85 90 95 Val Ser Lys Thr Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu 100 105 110 Leu Thr Val Ala Leu His Tyr Val Phe Asn Thr Pro Gln Asp Lys Leu 115 120 125 Leu Trp Asp Val Gly His Gln Ser Tyr Pro His Lys Ile Leu Thr Gly 130 135 140 Arg Arg Asp Lys Met Pro Thr Met Arg Gln Thr Asn Gly Leu Ser Gly 145 150 155 160 Phe Val Lys Arg Ser Glu Ser Glu Tyr Asp Ser Phe Gly Thr Gly His 165 170 175 Ser Ser Thr Thr Ile Ser Ala Ala Leu Gly Met Ala Val Gly Arg Asp 180 185 190 Leu Lys Gly Ala Lys Asn Asn Val Val Ala Val Ile Gly Asp Gly Ala 195 200 205 Met Thr Ala Gly Gln Ala Tyr Glu Ala Met Asn Asn Ala Gly Tyr Leu 210 215 220 Asp Ser Asp Met Ile Val Ile Leu Asn Asp Asn Lys Gln Val Ser Leu 225 230 235 240 Pro Thr Ala Thr Leu Asp Gly Pro Ala Pro Pro Val Gly Ala Leu Ser 245 250 255 Gly Ala Leu Ser Lys Leu Gln Ser Ser Arg Pro Leu Arg Glu Leu Arg 260 265 270 Glu Val Ala Lys Gly Val Thr Lys Gln Ile Gly Gly Ser Val His Glu 275 280 285 Ile Ala Ala Lys Val Asp Glu Tyr Ala Arg Gly Met Ile Ser Gly Ser 290 295 300 Gly Ser Ser Leu Phe Glu Glu Leu Gly Leu Tyr Tyr Ile Gly Pro Val 305 310 315 320 Asp Gly His Asn Ile Asp Asp Leu Ile Thr Ile Leu Arg Glu Val Lys 325 330 335 Gly Thr Lys Thr Thr Gly Pro Val Leu Ile His Val Ile Thr Glu Lys 340 345 350 Gly Arg Gly Tyr Pro Tyr Ala Glu Arg Ala Ser Asp Lys Tyr Gln Arg 355 360 365 Val Ala Lys Phe Asp Pro Ala Thr Gly Arg Gln Phe Lys Gly Pro Ala 370 375 380 Lys Thr Pro Ser Tyr Asn Asn Tyr Phe Ala Glu Pro Leu Ile Ala Gln 385 390 395 400 Ala Gly Gln Asp Ser Lys Ile Val Ala Phe His Pro Ala Met Gly Gly 405 410 415 Gly Thr Gly Leu Asn Tyr Phe Leu Arg Arg Phe Pro Asn Arg Gly Phe 420 425 430 Gln Val Glu Ser Ala Lys Gln Asn Ala Val Thr Leu Pro Gly Arg Leu 435 440 445 Ala Gly Lys Gly Val Lys Pro Phe Cys Ala 450 455 33 719 PRT Capsicum annuum 33 Met Ala Leu Cys Ala Tyr Ala Phe Pro Gly Ile Leu Asn Arg Thr Val 1 5 10 15 Ala Val Ala Ser Asp Ala Ser Lys Pro Thr Pro Leu Phe Ser Glu Trp 20 25 30 Ile His Gly Thr Asp Leu Gln Phe Gln Phe His Gln Lys Leu Thr Gln 35 40 45 Val Lys Lys Arg Ser Arg Thr Val Gln Ala Ser Leu Ser Glu Ser Gly 50 55 60 Glu Tyr Tyr Thr Gln Arg Pro Pro Thr Pro Ile Val Asp Thr Ile Asn 65 70 75 80 Tyr Pro Ile His Met Lys Asn Leu Ser Leu Lys Glu Leu Lys Gln Leu 85 90 95 Ala Asp Glu Leu Arg Ser Asp Thr Ile Phe Asn Val Ser Lys Thr Gly 100 105 110 Gly His Leu Gly Ser Ser Leu Gly Val Val Glu Leu Thr Val Ala Leu 115 120 125 His Tyr Val Phe Asn Ala Pro Gln Asp Arg Ile Leu Trp Asp Val Gly 130 135 140 His Gln Ser Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Glu Lys Met 145 150 155 160 Ser Thr Leu Arg Gln Thr Asn Gly Leu Ala Gly Phe Thr Lys Arg Ser 165 170 175 Glu Ser Glu Tyr Asp Cys Phe Gly Thr Gly His Ser Ser Thr Thr Ile 180 185 190 Ser Ala Gly Leu Gly Met Ala Val Gly Arg Asp Leu Lys Gly Arg Asn 195 200 205 Asn Asn Val Ile Ala Val Ile Gly Asp Gly Ala Met Thr Ala Gly Gln 210 215 220 Ala Tyr Glu Ala Met Asn Asn Ala Gly Tyr Leu Asp Ser Asp Met Ile 225 230 235 240 Val Ile Leu Asn Asp Asn Arg Gln Val Ser Leu Pro Thr Ala Thr Leu 245 250 255 Asp Gly Pro Val Pro Pro Val Gly Ala Leu Ser Ser Ala Leu Ser Arg 260 265 270 Leu Gln Ser Asn Arg Pro Leu Arg Glu Leu Arg Glu Val Ala Lys Gly 275 280 285 Val Thr Lys Gln Ile Gly Gly Pro Met His Glu Leu Ala Ala Lys Val 290 295 300 Asp Glu Tyr Ala Arg Gly Met Ile Ser Gly Ser Gly Ser Thr Leu Phe 305 310 315 320 Glu Glu Leu Gly Leu Tyr Tyr Ile Gly Pro Val Asp Gly His Asn Ile 325 330 335 Asp Asp Leu Ile Ser Ile Leu Lys Glu Val Arg Ser Thr Lys Thr Thr 340 345 350 Gly Pro Val Leu Ile His Val Val Thr Glu Lys Gly Arg Gly Tyr Pro 355 360 365 Tyr Ala Glu Arg Ala Ala Asp Lys Tyr His Gly Val Ala Lys Phe Asp 370 375 380 Pro Ala Thr Gly Lys Gln Phe Lys Gly Ser Ala Lys Thr Gln Ser Tyr 385 390 395 400 Thr Thr Tyr Phe Ala Glu Ala Leu Ile Ala Glu Ala Glu Ala Asp Lys 405 410 415 Asp Ile Val Ala Ile His Ala Ala Met Gly Gly Gly Thr Gly Met Asn 420 425 430 Leu Phe Leu Arg Arg Phe Pro Thr Arg Cys Phe Asp Val Gly Ile Ala 435 440 445 Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Cys Glu Gly Leu 450 455 460 Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Met Gln Arg Ala Tyr Asp 465 470 475 480 Gln Val Val His Asp Val Asp Leu Gln Lys Leu Pro Val Arg Phe Ala 485 490 495 Met Asp Arg Ala Gly Leu Val Gly Ala Asp Gly Pro Thr His Cys Gly 500 505 510 Ala Phe Asp Val Thr Phe Met Ala Cys Leu Pro Asn Met Val Val Met 515 520 525 Ala Pro Ser Asp Glu Ala Glu Leu Phe His Ile Val Ala Thr Ala Ala 530 535 540 Ala Ile Asp Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly Asn Gly 545 550 555 560 Ile Gly Val Glu Leu Pro Ala Gly Asn Lys Gly Ile Pro Leu Glu Val 565 570 575 Gly Lys Gly Arg Ile Leu Val Glu Gly Glu Arg Val Ala Leu Leu Gly 580 585 590 Tyr Gly Ser Ala Val Gln Asn Cys Leu Ala Ala Ala Ser Val Leu Glu 595 600 605 Ser Arg Gly Leu Gln Val Thr Val Ala Asp Ala Arg Phe Cys Lys Pro 610 615 620 Leu Asp Arg Ala Leu Ile Arg Ser Leu Ala Lys Ser His Glu Val Leu 625 630 635 640 Val Thr Val Glu Lys Gly Ser Ile Gly Gly Phe Gly Ser His Val Val 645 650 655 Gln Phe Met Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys Trp Arg 660 665 670 Pro Ile Val Leu Pro Asp Arg Tyr Ile Asp His Gly Ser Pro Ala Asp 675 680 685 Gln Leu Ala Glu Ala Gly Leu Thr Pro Ser His Ile Ala Ala Thr Val 690 695 700 Phe Asn Ile Leu Gly Gln Thr Arg Glu Ala Leu Glu Val Met Thr 705 710 715 34 594 PRT Oryza sativa 34 Asn Tyr Pro Ile His Met Lys Asn Leu Ser Leu Lys Glu Leu Gln Gln 1 5 10 15 Leu Ala Asp Glu Leu Arg Ser Asp Val Ile Phe His Val Ser Lys Thr 20 25 30 Gly Gly His Leu Gly Ser Ser Leu Gly Val Val Glu Leu Thr Val Ala 35 40 45 Leu His Tyr Val Phe Asn Thr Pro Gln Asp Lys Ile Leu Trp Asp Val 50 55 60 Gly His Gln Ser Tyr Pro His Lys Ile Leu Thr Gly Arg Arg Asp Lys 65 70 75 80 Met Pro Thr Met Arg Gln Thr Asn Gly Leu Ser Gly Phe Thr Lys Arg 85 90 95 Ser Glu Ser Glu Tyr Asp Ser Phe Gly Thr Gly His Ser Ser Thr Thr 100 105 110 Ile Ser Ala Ala Leu Gly Met Ala Val Gly Arg Asp Leu Lys Gly Gly 115 120 125 Lys Asn Asn Val Val Ala Val Ile Gly Asp Gly Ala Met Thr Ala Gly 130 135 140 Gln Ala Tyr Glu Ala Met Asn Asn Ala Gly Tyr Leu Asp Ser Asp Met 145 150 155 160 Ile Val Ile Leu Asn Asp Asn Lys Gln Val Ser Leu Pro Thr Ala Thr 165 170 175 Leu Asp Gly Pro Ala Pro Pro Val Gly Ala Leu Ser Ser Ala Leu Ser 180 185 190 Lys Leu Gln Ser Ser Arg Pro Leu Arg Glu Leu Arg Glu Val Ala Lys 195 200 205 Gly Val Thr Lys Gln Ile Gly Gly Ser Val His Glu Leu Ala Ala Lys 210 215 220 Val Asp Glu Tyr Ala Arg Gly Met Ile Ser Gly Ser Gly Ser Thr Leu 225 230 235 240 Phe Glu Glu Leu Gly Leu Tyr Tyr Ile Gly Pro Val Asp Gly His Asn 245 250 255 Ile Asp Asp Leu Ile Thr Ile Leu Arg Glu Val Lys Ser Thr Lys Thr 260 265 270 Thr Gly Pro Val Leu Ile His Val Val Thr Glu Lys Gly Arg Gly Tyr 275 280 285 Pro Tyr Ala Glu Arg Ala Ala Asp Lys Tyr His Gly Val Ala Lys Phe 290 295 300 Asp Pro Ala Thr Gly Lys Gln Phe Lys Ser Pro Ala Lys Thr Leu Ser 305 310 315 320 Tyr Thr Asn Tyr Phe Ala Glu Ala Leu Ile Ala Glu Ala Glu Gln Asp 325 330 335 Asn Arg Val Val Ala Ile His Ala Ala Met Gly Gly Gly Thr Gly Leu 340 345 350 Asn Tyr Phe Leu Arg Arg Phe Pro Asn Arg Cys Phe Asp Val Gly Ile 355 360 365 Ala Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Cys Glu Gly 370 375 380 Leu Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Leu Gln Arg Gly Tyr 385 390 395 400 Asp Gln Val Val His Asp Val Asp Leu Gln Lys Leu Pro Val Arg Phe 405 410 415 Ala Met Asp Arg Ala Gly Leu Val Gly Ala Asp Gly Pro Thr His Cys 420 425 430 Gly Ala Phe Asp Val Thr Tyr Met Ala Cys Leu Pro Asn Met Val Val 435 440 445 Met Ala Pro Ser Asp Glu Ala Glu Leu Cys His Met Val Ala Thr Ala 450 455 460 Ala Ala Ile Asp Asp Arg Pro Ser Cys Phe Arg Tyr Pro Arg Gly Asn 465 470 475 480 Gly Ile Gly Val Pro Leu Pro Pro Asn Tyr Lys Gly Val Pro Leu Glu 485 490 495 Val Gly Lys Gly Arg Val Leu Leu Glu Gly Glu Arg Val Ala Leu Leu 500 505 510 Gly Tyr Gly Ser Ala Val Gln Tyr Cys Leu Ala Ala Ala Ser Leu Val 515 520 525 Glu Arg His Gly Leu Lys Val Thr Val Ala Asp Ala Arg Phe Cys Lys 530 535 540 Pro Leu Asp Gln Thr Leu Ile Arg Arg Leu Ala Ser Ser His Glu Val 545 550 555 560 Leu Leu Thr Val Glu Glu Gly Ser Ile Gly Gly Phe Gly Ser His Val 565 570 575 Ala Gln Phe Met Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys Trp 580 585 590 Arg Pro 

What is claimed is:
 1. An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having 1-deoxy-D-xylulose 5-phosphate synthase activity, wherein the polypeptide has an amino acid sequence of at least 95% sequence identity, based on the Clustal method of alignment, when compared to SEQ ID NO:10.
 2. The polynucleotide of claim 1 wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:10.
 3. The polynucleotide of claim 1, wherein the nucleotide sequence of the polynucleotide comprises SEQ ID NO:9.
 4. An isolated complement of the polynucleotide of claim 1, wherein (a) the complement and the nucleotide sequence of the polynucleotide consist of the same number of nucleotides, and (b) the nucleotide sequences of the complement and the polynucleotide have 100% complementarity.
 5. A cell or a virus comprising the polynucleotide of claim
 1. 6. The cell of claim 5, wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell, an insect cell, and a plant cell.
 7. A method for transforming a cell comprising introducing into a cell the polynucleotide of claim
 1. 8. A method for producing a transgenic plant comprising (a) transforming a plant cell with the polynucleotide of claim 1, and (b) regenerating a plant from the transformed plant cell.
 9. A method for isolating a polypeptide having 1-deoxy-D-xylulose 5-phosphate synthase activity comprising isolating the polypeptide from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.
 10. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.
 11. The recombinant DNA construct of claim 10, wherein the recombinant DNA construct is an expression vector.
 12. A method for altering the level of 1-deoxy-D-xylulose 5-phosphate synthase polypeptide expression in a host cell, the method comprising: (a) Transforming a host cell with the recombinant DNA construct of claim 10; and (b) Growing the transformed cell in step (a) under conditions suitable for the expression of the recombinant DNA construct.
 13. A method for evaluating at least one compound for its ability to inhibit 1-deoxy-D-xylulose 5-phosphate synthase activity, comprising the steps of: (a) introducing into a host cell the recombinant DNA construct of claim 10; (b) growing the host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of a 1-deoxy-D-xylulose 5-phosphate synthase; (c) optionally purifying the 1-deoxy-D-xylulose 5-phosphate synthase from the host cell; (d) treating the 1-deoxy-D-xylulose 5-phosphate synthase with a compound to be tested; (e) comparing the activity of the 1-deoxy-D-xylulose 5-phosphate synthase that has been treated with a test compound to the activity of an untreated 1-deoxy-D-xylulose 5-phosphate synthase, and selecting compounds with potential for inhibitory activity.
 14. A plant comprising the recombinant DNA construct of claim
 10. 15. A seed comprising the recombinant DNA construct of claim
 10. 16. An isolated polypeptide having 1-deoxy-D-xylulose 5-phosphate synthase activity, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:10.
 17. An isolated polypeptide having 1-deoxy-D-xylulose 5-phosphate synthase activity, wherein the amino acid sequence of the polypeptide has an amino acid sequence of at least 95% sequence identity, based on the Clustal method of alignment, when compared to SEQ ID NO:10. 